WO2009092808A1

WO2009092808A1 - Method for detecting phosphopantetheinyl transferase activity

Info

Publication number: WO2009092808A1
Application number: PCT/EP2009/050799
Authority: WO
Inventors: Sean Doyle; Deirdre Stack
Original assignee: National University Of Ireland, Maynooth
Priority date: 2008-01-24
Filing date: 2009-01-23
Publication date: 2009-07-30

Abstract

The invention provides a method for detecting 4 -phosphopantetheinyl transferase (4 PPTase) activity, the method comprising the steps of: (a) immobilising a carrier protein domain or a functional fragment thereof on a solid support suitable for this purpose; (b) reacting the immobilised carrier protein domain or functional fragment thereof of step (a) with labelled coenzyme A and 4' PPTase to form a labelled carrier protein or functional fragment thereof; and (c) detecting the presence of the labelled carrier protein domain or functional fragment thereof.

Description

Method for detecting phosphopantetheinyl transferase activity

The present invention relates to a method for detecting phosphopantetheinyl transferase (4'PPTase) activity. The present invention also relates to a method for the indirect labelling of a non-native protein during protein phosphopantetheinylation.

4 '-phosphopantetheinyl transferases (4'PPTases) are enzymes which catalyse the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to the carrier protein domain of certain enzymes, including enzymes involved in nonribosomal peptide, polyketide and fatty acid biosynthesis, for activation of these enzymes. This enzyme activation, known as protein phosphopantetheinylation, is essential for the growth of microorganisms.

Nonribosomal peptide (NRP) synthetases are multi-functional enzymes composed of distinct functional domains (adenylation (A), thiolation (T or peptidyl carrier protein (PCP)) and condensation (C) domains) which when grouped together are referred to as a 'module'. Thiolation domains are also referred to as peptidyl carrier protein (PCP) domains and therefore the acronyms T and PCP are interchangeably used. Additionally, other enzyme functions are occasionally present in NRP synthetases, including epimerase (E) and thioesterase (TE) domains which are responsible for L- to D-amino acid conversion and release of the newly-synthesised nonribosomal peptide from the NRP synthetase, respectively. An NRP synthetase can contain one or more modules and each module within an NRP synthetase is responsible for recognising (via the A domain) and incorporating one amino acid (or nonproteogenic amino acid) into nonribosomal peptide product.

NRP synthetases are produced in the inactive (apo -enzyme) form and require post- translational modification to become functionally active. Post-translational modification of NRP synthetases requires transfer of a 4'-phosphopantetheine group, derived from coenzyme A, to a specific serine residue within the thiolation domain(s) of the NRP synthetase and this reaction is catalysed by a 4'-phosphopantetheinyl transferase. The 4'- phosphopantetheine arm on T domains can then transfer covalently bound amino acids (recognized and activated by the A domain) to the condensation domain where peptide bond formation occurs between adjacent tethered amino acids. Therefore the T domain is involved in the overall enzymatic function of the NRP synthetase. To date, the protein phosphopantetheinylation reaction has been investigated using complicated, time-consuming and expensive solution phase assays. For example, the efficacy of novel anthranilic acid inhibitors of the B. subtilis acyl carrier protein synthase (AcpS), which catalyses the transfer of 4'phosphopantetheinyl group from CoA onto a conserved serine residue present on an Escherichia coli acyl carrier protein domain has been investigated using a solution phase assay (Joseph-Mc Carthy et al, 2005, J.Med. Chem. 48(25), 7960-7969). The solution phase assay involved incubation of the AcpS 4'PPTase enzyme in the presence of a GST-linked ACP carrier protein and a biotin- CoA reporter analogue at room temperature. Detection of the biotin labelled ACP was achieved by an overnight incubation with streptavidin-allophycocyanin conjugate followed by time-resolved fluorescence detection.

The above-mentioned disadvantages of conventional assays have limited the application of protein phosphopantetheinylation. In particular, these disadvantages have significantly limited the potential for high-throughput operations involving protein phosphopantetheinylation.

It is an object of the present invention to mitigate or eliminate the disadvantages associated with conventional methods of investigating protein phosphopantetheinylation.

It is also an object of the present invention to provide a method for investigating protein phosphopantetheinylation and detecting 4'PPTase activity which is simple, fast, high- throughput and cost-efficient.

It is a further object of the invention to provide a method for the indirect labelling of a non- native protein during protein phosphopantetheinylation.

It is a still further object of the present invention to provide a method for detecting 4'PPTase activity which can be used for screening libraries of candidate compounds as inhibitors of 4'PPTase.

According to the invention there is provided a method for detecting 4'- phosphopantetheinyl transferase (4'PPTase) activity, the method comprising the steps of: (a) immobilising a carrier protein domain or a functional fragment thereof on a solid support suitable for this purpose;

(b) reacting the immobilised carrier protein domain or functional fragment thereof of step (a) with labelled coenzyme A and 4'PPTase to form a labelled carrier protein domain or functional fragment thereof; and

(c) detecting the presence of the labelled carrier protein domain or functional fragment thereof.

The invention also provides a method for detecting 4'-phosphopantetheinyl transferase (4'PPTase) activity, the method comprising the steps of:

(a)(i) reacting a carrier protein domain or a functional fragment thereof with labelled coenzyme A and 4'PPTase to form a labelled carrier protein domain or functional fragment thereof;

(b)(i) immobilising the labelled carrier protein domain or functional fragment thereof of step (a)(i) on a solid support suitable for this purpose via the carrier protein domain or functional fragment thereof; and

(c)(i) detecting the presence of the labelled carrier protein domain or functional fragment thereof.

The invention further provides a method for the indirect labelling of a non-native protein, the method comprising the steps of:

(a) providing a fusion protein comprising a carrier protein domain or a functional fragment thereof and a non-native protein fused thereto;

(b) immobilising the fusion protein on a solid support suitable for this purpose via the carrier protein domain or functional fragment thereof;

(c) reacting the immobilised fusion protein of step (b) with labelled coenzyme A and 4'PPTase to form a labelled carrier protein domain or functional fragment thereof, thereby indirectly labelling the non-native protein; and optionally

(d) detecting the presence of the labelled non-native protein by detecting the presence of the labelled carrier protein domain or functional fragment thereof.

The invention still further provides a method for the indirect labelling of a non-native protein, the method comprising the steps of: (a)(i) providing a fusion protein comprising a carrier protein domain or a functional fragment thereof and a non-native protein fused thereto;

(b)(i) reacting the fusion protein with labelled coenzyme A and 4'PPTase to form a labelled fusion protein labelled via the carrier protein domain or functional fragment thereof, thereby indirectly labelling the non-native protein;

(c)(i) immobilising the labelled fusion protein of step (b)(i) on a solid support suitable for this purpose via the labelled carrier protein domain or functional fragment thereof; and optionally

(d)(i) detecting the presence of the labelled non-native protein by detecting the presence of the labelled carrier protein domain or functional fragment thereof.

As used herein, the term "functional fragment" is intended to mean the minimum number, or sequence, of amino acids which should be, or need to be, present to allow the carrier protein domain to function as an acceptor of a 4'-phosphopantetheine arm.

The step of detecting the presence of the labelled carrier protein domain or functional fragment thereof may also comprise measuring the amount of labelled carrier protein domain or functional fragment thereof formed. Accordingly, the methods of the invention may involve qualitative and quantitative detection of the labelled carrier protein domain or functional fragment thereof.

The carrier protein domain or functional fragment thereof may be derived from or may be a component of an enzyme selected from a nonribosomal peptide (NRP) synthetase, a fatty acid synthetase and a polyketide synthetase, preferably an NRP synthetase. The carrier protein domain or functional fragment thereof may be an acyl carrier protein (ACP) domain or a functional fragment thereof, or a peptidyl carrier protein (PCP) domain or a functional fragment thereof, preferably a PCP domain. In a particularly preferred embodiment, the carrier protein domain or functional fragment thereof is a PCP domain derived from or which is a component of an NRP synthetase. Thus, an enzyme e.g. an NRP synthetase may be used in the invention, wherein the PCP domain component of the enzyme will be immobilised in accordance with the invention. As an alternative to including the whole enzyme, the PCP domain may be present in the form of an AT di- domain, wherein A represents an adenylation domain and T represents the thiolation/PCP domain. Consequently, the invention may also comprise immobilising an AT di-domain on the solid support via the T domain. The carrier protein domain or functional fragment thereof, whether labelled or not, may be immobilised on the solid support by covalent attachment or passive adsorption, possibly involving hydrophobic interactions. The carrier protein domains or functional fragments thereof may be obtained recombinantly or by chemical synthesis.

The enzyme from which the carrier protein domain or functional fragment thereof may be derived or is a component of, is preferably derived from a microorganism, such as a fungus or bacterium. The fungus may be selected from the group comprising Aspergillus fumigatus, Aspergillus nidulans, Cochliobolus heterostrophus, Trichoderma virens, Magnaporthe grisea, Trichoderma harzianum, Claviceps purpurea, Gibberella zeae, Fusarium graminearum and Penicillium spp, more preferably Aspergillus fumigatus. The bacterium may be selected from the group comprising Pseudomonas spp., Mycobacterium tuberculosis, Angiococcus disciformis, Myxococcus xanthus, Xanthomonas albilineans, Streptomyces spp, and cyanobacteria. In a particularly preferred embodiment, the carrier protein domain or functional fragment thereof is a PCP domain derived from or which is a component of an NRP synthetase which is in turn derived from Aspergillus fumigatus, as presented in SEQ ID NOS. 1 and 2.

The 4'PPT ase is preferably derived from Aspergillus fumigatus, as presented in SEQ ID NOS. 20 and 21. The 4'PPTase may be obtained from suitable host cells such as bacterial cells, yeast cells, mammalian cells, plant cells and insect cells, preferably recombinant insect cells, more preferably recombinant Spodoptera frugiperdag (Sfg) insect cells.

The carrier protein domain or functional fragment thereof may have a non-native protein fused thereto.

As used herein, the term "non-native protein" is intended to mean a protein which would not normally be linked to the carrier protein domain or functional fragment thereof in nature.

When the carrier protein domain or functional fragment thereof has a non-native protein fused thereto, the methods of the invention may conveniently comprise indirectly labelling the non-native protein by reacting the carrier protein domain with labelled coenzyme A and 4'PPTase to form a labelled carrier protein domain or functional fragment thereof. Furthermore, when the carrier protein domain or functional fragment thereof has a non- native protein fused thereto, the presence of the labelled non-native protein may be detected by detecting the presence of the labelled carrier protein domain or functional fragment thereof. Similarly, a native protein of which the carrier protein domain or functional fragment thereof is a component may be labelled and/or detected via the carrier protein domain or functional fragment thereof.

The non-native protein is preferably selected from the group comprising antibodies, antigens, enzymes and receptors. The non-native protein is conveniently an enzyme, such as a glutathione ^-transferase (GST), preferably a GST derived from Aspergillus fumigatus, as presented in SEQ ID NOS. 22 and 23. When a fusion protein comprising a carrier protein or a functional fragment thereof and a non-native protein fused thereto is used in the method of the invention, the fusion protein is preferably a recombinant fusion protein. A PCP-GST fusion protein, also called a T-GST fusion protein, is preferred, as presented in SEQ ID NOS. 24 and 25.

The coenzyme A (CoA) may be labelled with biotin, fluorescein, a protein, a single- or double-stranded nucleic acid, or a carbohydrate. Preferably, the coenzyme A is labelled with biotin. When the label is a protein, it is preferably selected from the group comprising antibodies, antigens, enzymes and receptors.

The label is attached to coenzyme A via the thiol group of the phosphopantetheine arm. Upon reaction with 4'PPT ase, the phosphopantetheine arm and the attached label will be transferred to the carrier protein domain or functional fragment thereof which is immobilised on the solid support either before or after the reaction with 4'PPTase. Accordingly, this provides a mechanism for immobilising the label indirectly to the solid support and would overcome any problems that might be associated with direct immobilisation of same, such as lack of binding to, or affinity for, the solid support.

The solid support may comprise a plastics material, glass or metal. Alternatively, the solid support may comprise a combination of a plastics material or glass with metal, such as metal-coated plastics material or metal-coated glass. Suitable plastics materials include polymers such as polystyrene, polyethylene, polypropylene, polytetrafluoroethylene, polyamide, polyacrylamide and polyvinylchloride, optionally in microparticulate form. Polymers sold under the trade name Nunc MaxiSorb are preferred. Suitable metals include gold, silver, aluminium, chromium and titanium, optionally in microparticulate or nanoparticulate form.

Prior to reacting the immobilised carrier protein domain or functional fragment thereof with labelled coenzyme A and 4'PPTase to form a labelled carrier protein domain or functional fragment thereof, a blocking agent may be used to prevent non-specific binding of the 4'PPTase to the solid support or carrier protein domain or functional fragment thereof and/or to stabilise the immobilised carrier protein domain or functional fragment thereof. Suitable blocking agents include bovine serum albumin (BSA), gelatin and casein, preferably BSA.

The invention further provides a kit for use in the methods of the invention, comprising: (a) a solid support;

(b) a carrier protein domain or a functional fragment thereof for immobilising on, or immobilised on, the support;

(c) labelled coenzyme A; and

(d) 4'PPTase; wherein the components listed in (a) - (d) are as defined above.

The kit may also comprise:

(e) means for detecting the presence of the labelled carrier protein domain or functional fragment thereof, for example tetramethylbenzidine (TMB) for colorimetric detection of biotin-labelled carrier protein domain or functional fragment thereof;

(f) a suitable buffer; and

(g) instructions for use.

The carrier protein or functional fragment thereof forming part of the kit may comprise a non-native protein fused thereto.

The methods of the invention may be applied to the screening of libraries of candidate compounds for 4'PPTase inhibition for use in the development of antimicrobial drugs. The candidate compounds may be introduced into the reaction medium with the 4'PPTase, either with or without pre-incubation with the 4'PPTase. If the candidate compounds are pre-incubated with 4'PPTase, they may be pre-incubated with or without CoA. 4'PPTase inhibition will result in reduced labelling of carrier protein domains or functional fragments thereof.

Advantages of the invention include the following:

• It provides a simple, fast, high-throughput and cost-efficient assay and kit for detecting 4'PPTase activity.

• It allows the indirect immobilisation of the label on the solid support thereby avoiding any problems encountered with direct immobilisation of same.

• It minimises interference in the detecting step from unreacted components such as unreacted labelled CoA which, as a result of the invention, will not bind to the solid support.

• It allows labelling of any protein of which the carrier protein domain is a component. Thus, it conveniently allows labelling of e.g. the enzyme NRP synthetase, or a component thereof e.g. an AT di-domain, wherein A represents an adenylation domain and T represents the thiolation/PCP domain, by labelling the T domain in accordance with the methods of the invention.

• Labelling of an AT di-domain using the methods of the invention would conveniently provide confirmation of the functionality of the AT di-domain which could then be used for subsequent investigations. For example, the AT di-domain could subsequently be used in an assay to determine which amino acids are recognized and activated by A domains. More specifically, if a test aliquot removed from an apo-AT di-domain preparation was confirmed to be labelled with biotin or fluorescein, according to the method of the invention, then labelling of the remainder of the apo-AT di-domain preparation with 4'-phosphopantetheine derived from unlabelled CoA, via 4'PPTase activity, would be confirmed. This latter form could then be used for the study of the A domain ability to recognise a specific amino acid for NRPS. • It allows generic protein labelling since carrier protein domains or functional fragments thereof still function when attached to non-native protein thereby extending the potential use of carrier protein domains or functional fragments thereof as labelling vehicles for other proteins. • It may be applied to the screening of libraries of candidate compounds for 4'PPTase inhibition for use in the development of antimicrobial drugs.

• It provides a facile high-throughput strategy for the activity determination of 4'PPTase in the development of inhibitors of 4'PPTase activity as antimicrobial drugs, and for "green chemistry" approaches to chemical biosyntheses.

• It supercedes existing approaches by using a PCP (thiolation) domain to enable recombinant protein labelling.

• It surprisingly overcomes conventional problems associated with the direct labelling of proteins, e.g. inactivation due to conformational restrictions. • It provides an affinity tag on non-essential portions of recombinant proteins to aid purification.

• It presents a defined combination of reagents which can be used for protein labelling.

The following examples serve to illustrate the invention but it will be appreciated that the invention is not limited to these examples.

EXAMPLES

MATERIALS AND METHODS

2.1. Bioinformatic analysis.

Preliminary sequence data was obtained from The Institute for Genomic Research website at http ://www.tigr.org. The unannotated genome of Aspergillus fumigatus (A. fumigatus ATCC293) was initially interrogated to identify open reading frames corresponding to novel nonribosomal peptide synthetases. One open reading frame, which encoded a putative nonribosomal peptide synthetase, was identified, and was subsequently termed pes3 (SEQ ID NOS. 5 and 6, taken from http://www.cadre-genomes.org.uk; Figure IA). Sequence homologies were determined using the BLAST algorithm [http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html] and ClustalW [http://www.ebi.ac .uk/clustalw/1. For the purpose of this specification, the nomenclature pes3 is synonymous with NRPS8 (Cramer R.A. Jr et al, (2006), Phylogenomic analysis of non-ribosomal peptide synthetases in the genus Aspergillus Gene 383, 24-32) and Afu5gl2730 (Mabey et al., (2004). CADRE: the Central Aspergillus Data Repository. Nucleic Acids Res 32, 401-405 and Merman et al, (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151- 1156). The/?es3^r amplicon, which is derived from the 5' end of the entire pes3 gene, and which includes an adenylation (A) domain and a thiolation (T or PCP) domain, is presented in SEQ ID NOS. 3 and 4. The region encoding the thiolation (PCP) domain derived &ompes3Λτ, is presented in SEQ ID NOS. 1 and 2. Further information concerning bioinformatic analysis and the sequences generally, including sequence alignments, is provided below and also in Section 3.6.

2.2. Fungal DNA isolation and PCR amplification.

A. fumigatus strain ATCC 26933 was grown (250 ml cultures) at 37⁰C in Minimal Essential Medium (MEM) (5%(v/v) containing Fetal Calf Serum (FCS)) for 2 days. Genomic DNA was isolated by crushing fungal mycelia in liquid N₂ followed by phenolxhloroform extraction, washing with 70%(v/v) ethanol and final DNA resuspension in 10 mM Tris-HCl, 1 mM EDTA pH 8.0 (200 μl). PCR was carried out using AccuTaq LA polymerase (Sigma) using a total of 5 ng of genomic DNA per PCR reaction. PCR reactions also included 1OX AccuTaq reaction buffer, 0.25 μM of each dNTP, 1.0 μM forward and reverse primer in a total volume of 20 μl, as shown in Table 1.

Gene (F/R) Primer Sequences (5'-3') Amplicon

(bp)

^"JeIJJT F GA^AITCG^G^CA^AC^^

1800 (SEQ ID NO. 7) pes3_ΛT R GAGAAAGCTTAGTGATCGAGGAACGCAA (SEQ ID NO. 8)

T/PCP F GAGAGGATCCCATGGAACGCCGGTTGCGAG

196 (SEQ ID NO. 9)

T/PCP R GAGAGAATTCTGCGAGCTCTGTGATGGA

(SEQ ID NO. 10) Table 1. Oligonucleotide primers designed for directional cloning of pes3_ΛT and T/PCP region sequences into the pProEx-GstB expression vector.

PCR reactions comprised an initial step of 60 s denaturation at 95°C, followed by 30 cycles consisting of 60 s at 95°C, 30 s at 55°C, 90 s at 72°C and finally for 360 s at 72°C. Amplicons were analysed by agarose gel electrophoresis whereby 5 μl of product was electrophoresed on 1% (w/v) agarose containing 0.5 μg/ml of ethidium bromide for 30 min at 100 V. Relevant amplicons were visualised using an 'Eagle-Eye IF digital still video system (Stratagene, CA, USA). DNA sequence analysis was carried out on a commercial basis by MWG Biotech (Germany).

2.3. Cloning and expression of the nonribosomal peptide synthetase gene fragment, pes3_Aτ.

The pes 3 _AT Αmplicon, which is derived from the 5' end of the entire pes 3 gene (Figure IA) was initially cloned into the TOPO® cloning vector and subsequently cloned into pProEx™ expression vectors (Invitrogen) according to the manufacturer's instructions. pFroΕx:pes3Aτ, the expression vector containing A. fumigatus pes3 AT , was transformed into E. coli according to the manufacturer's guidelines (Invitrogen). Protein expression was induced by the addition of isopropyl β-D-thiogalactoside (IPTG); 0.6 mM final concentration) and monitored by SDS-PAGE, Western blot analysis using monoclonal antibody reactivity against the HiS₆ fusion peptide (Figure 2) and MALDI-ToF mass spectrometry (Table 4).

2.4. Recombinant PCS3_AT purification and refolding. Recombinant Pes3Aτ was purified as follows: E. coli cells were lysed in the presence of protease inhibitors (phenylmethylsulfonyl fluoride (0.1 mM), pepstatin (2 μg/ml) and leupeptin (2 μg/ml)) by the addition of phosphate-buffered saline-sodium deoxycholate (0.5%(w/v)), subjected to DNAse (Sigma, Poole, UK) treatment (final concentration: 10 μg/ml) and the insoluble pellet washed extensively to remove contaminating proteins. Insoluble Pes3Aτ was resuspended in Guanidine-HCl (0.5-1.0 ml, 6 M), containing dithiothreitol (5 mM). Purified recombinant Pes3Aτ (250 μg/ml) in 50 mM sodium carbonate pH 9.4 containing 6 M Guanidine-HCL was serially dialysed into 5OmM sodium carbonate pH 9.4 containing 8 M Urea, then 4 M Urea, followed by dialysis into 5OmM sodium carbonate buffer pH 9.4 to facilitate solubilisation.

2.5. 4'PPTase extraction from infected (Sf₉) insect cells.

Tissue culture flasks (75-mm, Sarstedt) were seeded with 9.0 x 10⁶ Sf ₉ insect cells (available from Invitrogen (http://www.invitrogen.com) in 10 ml Grace's Complete medium and placed on a rocker at 27 ⁰C for 30 min. Cells were infected with recombinant baculovirus (encoding A. fumigatus 4'PPTase (SEQ ID NOS. 20 and 21)) at multiplicity of infection (MOI) of 10. The calculated volume of inoculum needed to infect each flask was added to Grace's Complete medium in a total volume of 1.2 ml. The medium was removed from the flasks and the virus was added, for 1 hour on a rocking platform. After this period Grace's Complete medium (10 ml) was added to the flasks followed by incubation for 4 days at 27 ⁰C. Cells were removed by hitting the flasks on their sides and pipette transferred to sterile tubes. The samples were centrifuged at 1000 x g for 20 min at 27 ⁰C, and cells were resuspended in PBS. The samples were analysed for recombinant protein expression via SDS-PAGE and Western blot (Figure 3). The Sf ₉ insect cells (5 x 10⁷) infected with recombinant baculovirus encoding 4'PPTase were resuspended in Ppant buffer (5 ml; 75 mM Tris-HCl, 5 mM Dithiothreitol (DTT) and 10 mM MgCl₂ pH 8.0) and sonicated (Bandelin Sonopuls, Progen Scientific Ltd, UK) for 3 x 5 sec at a maximum power for 5 min with 30 sec second intervals on ice. The resultant cell lysates were centrifuged at 10,000 x g for 10 min at 4 ⁰C. The supernatants were removed and analysed for the presence of 4'PPTase by Western blot analysis. The concentration of protein present in the cell lysate supernatants was determined by Bradford analysis and the supernatants divided into 0.5 ml aliquots and stored at -20⁰C for further use.

For purification of intracellular 4'PPTase, infected Sf ₉ insect cells (5 x 10⁷) were resuspended in lysis buffer (5 ml; 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole pH 8.0) containing 0.05%(w/v) sodium deoxycholate and sonicated (Bandelin Sonopuls, Progen Scientific Ltd, UK) for 3 x 5 sec at a maximum power for 5 min with 30 sec intervals on ice and the resultant lysates centrifuged at 10,000 x g for 10 min at 4 ⁰C. Purification proceeded under native conditions using NiNTA metal-chelate affinity chromatography Optimal protein yields were obtained using an optimised batch purification approach which entailed the incubation of the 4'PPTase lysate to the Ni-NTA resin for 2 hr with gentle inversion at 4 ⁰C. Subsequently, the protein:resin complex was packed into a column to facilitate washing and elution steps. Purified 4'PPTase was eluted with 250 mM imidazole and subsequently dialysed into Ppant buffer.

2.7. Coenzyme A analogue synthesis and solution phase 4'-PPTase activity analysis.

Coenzyme A (CoA) was covalently labelled through an available sulphydrl group by reaction with maleimido-biotin (MB) and -fluorescein (MFl) reagents according to La Clair et al. La Clair, J.J., Foley, T.L., Schegg, T.R., Regan, CM. and Burkart, M.D. (2004) Manipulation of carrier proteins in antibiotic biosynthesis. Chem. Biol. 11, 195- 201. . Reactions between MB/MF1 and CoA were allowed to proceed on ice for 30 min, followed by 10 min incubation at room temperature, and terminated by addition of IM DTT (1 μl). 4'-phosphopantetheinylation reactions using 4'-PPTase and recombinant Pes3Aτ were carried out as follows: Reaction mixtures for the in vitro phosphopantetheinylation assay contained Pes3Aτ (4.4 μM), 4' PPTase (0.08 μM), biotin-CoA (8.4 μM) in 75 mM Tris, 5 mM DTT and 10 mM MgCl₂, pH 8.0 (final volume: 100 μl). Reaction mixtures were incubated at 37⁰C for 1 hour. After incubation, reactions were terminated by the addition of 10%(w/v) trichloroacetic acid (900 μl) and centrifuged at 10,000 x g for 10 min. Pellets were re-suspended in IM Tris-HCl pH 8.0 and analysed by SDS-PAGE and Western blot analysis, using direct probing with Streptavidin-horseradish peroxidase and chemiluminescent substrate, to visualise biotinylated Pes3Aτ- Western blot detection was performed using streptavidin- horseradish peroxidase (HRP) conjugate (1/500) using ECL chemiluminescent detection (Pierce Biotechnology) (Figure 4a). Fluorescence detection was also undertaken by directly scanning SDS-PAGE gels, when fluorescein (Fl)-CoA was employed instead of biotin-CoA, using a Typhoon fluorescent scanner (GE Healthcare) at excitation and emission wavelength at 488/520 nm (Ex/Em) respectively (Figure 4b). Scanned gels were subsequently stained with Coomassie brilliant blue R (Figure 4c).

2.8 Microwell coating with recombinant Pes3Aτ for use in solid phase 4'phosphopantetheinylation assays.

In order to coat microtitre plates with PCP, microtitre plates were coated with purified recombinant Pes3Aτ (2 μg/ml) diluted into 50 mM sodium carbonate buffer pH 9.6 containing 0.01 % (w/v) sodium dodecyl sulphate (100 μl/well). Plates were incubated at 37°C for 2 hr and washed twice with PBST (200 μl). Blocking solution (l%(w/v) bovine serum albumin and 10%(w/v) sucrose in 50 mM sodium carbonate buffer pH 9.6; 200 μl/well) was applied to the wells and stored at 4°C overnight. Blocking solution was decanted off the plates and any residual solution was removed by tapping the plate upside down onto adsorbent paper and transferring the plate to a 37°C incubator for 1 hr prior to use. 2.9 Solid phase 4'phosphopantetheinylation using biotin-CoA

Reactants were prepared in triplicate in 1.5 ml microfuge tubes as outlined in Table 2.

Reagent stock solutions Purified 4'PPTase Control Biotin Buffer

4'PPTase crude cell cell CoA control lysate lysate control

μM)

4'PPTase Lysate (1.7 - 30 μl - - mg/ml Total cell protein)

Control Lysate (1.7 mg/ml - - 30 μl -

Total cell protein)

Biotin CoA (109 μm) 5 μl 5 μl 5 μl 5 μl

Ppant Buffer 55 μl 55 μl 95 μl lOOμl

Table 2. Reagent preparation for solid phase 4'phosphopantetheinylation assay (A). Final reagent concentrations; Biotin CoA (5.4 μM), 4'PPTase purified from infected Sfg cell supernatants (0.48 μM), 4'PPTase cell lysate obtained from infected Sf g insect cells (0.51 mg/ml total cell protein), and as a control, cell lysate obtained from un-infected Sf g insect cells, i.e. which contains no 4'PPTase (0.51 mg/ml total cell protein).

Aliquots (100 μl) of each sample were placed in wells of 96-well plates (Nunc maxisorb) which had been previously coated with Pes3Aτ (2 μg/ml), resulting in attachment of PCP to the well plates. Plates were incubated for 1 hr at room temperature to facilitate enzymatic reaction. Plates were then washed twice with PBST. Streptavidin - HRP (1/2000 dilution; 100 μl/ well) was added and plates incubated at 37°C for 1 hr. Plates were washed 4 times with PBST. Excess liquid was removed from the wells by tapping the plate out on adsorbent paper. Substrate tetramethylbenzidine (TMB) (100 μl/ well) was applied to the wells and left to develop for 10 min at room temperature. To terminate the reaction, IN H₂SO₄ (100 μl) was added to the wells and the absorbance values read at 450/630 nm using a plate reader (Bio-Tech, Synergy HT). The results are discussed in Section 3.3 below and are shown in Figure 6b.

2.10 Solid phase 4'phosphopantetheinylation using Fl-CoA

Reactants were prepared in triplicate in 1.5 ml microfuge tubes as outlined in Table 3. Reagents Purified 4'PPTase Control Fl- Buffer

4'PPTase crude cell cell CoA control lysate lysate control

Purified 4'PPTase (0.05μM) 95μl

4'PPTase Lysate (1.7 mg/ml - 30μl - - total cell protein)

Control Lysate (1.7mg/ml - - 30μl - total cell protein)

Fl- CoA (95 μm) 5μl 5μl 5μl 5μl

Ppant Buffer 60μl 60μl 90μl lOOμl

Table 3. Reagent preparation for solid phase 4'phosphopantetheinylation assay (B). Final reagent concentrations; Fl-CoA (3.6 μM), 4'PPTase purified from infected Sf g cell supernatants (0.48 μM), 4'PPTase cell lysate obtained from infected Sfg insect cells (0.51 mg/ml total cell protein), and as a control, cell lysate obtained from un-infected Sf g insect cells (0.51 mg/ml total cell protein).

Aliquots (100 μl) of each sample were placed in wells of black 96-well plates (Nunc maxisorb) which had been previously coated with Pes3Aτ (2 μg/ml), resulting in attachment of PCP to the well plates. Plates were incubated for 1 hr at room temperature in the dark while the enzymatic reaction occurred. Plates were then washed twice with PBST and excess liquid removed by tapping the plates on adsorbent paper. Fluorescence was detected using a plate reader (Bio-Tech, Synergy HT) with excitation and emission values of 490/520 nm respectively and fluorescence values recorded. The results are discussed in Section 3.3 below and are shown in Figure 6c.

2.11 Generation of recombinant fusion protein containing the Pes3AT derived thiolation domain.

The region encoding the thiolation (PCP) domain from pes3Aτ was fused to A.fumigatus glutathione ^-transferase B (gstB; Burns et al, 2005; SEQ ID NOS. 22 and 23) in order to assess the utility of the domain for non-native protein labelling (Figure IB). The fusion protein T-GstB is presented in SEQ ID NOS. 24 and 25. PCR cloning primers incorporated EcoRl & HmdIII restriction sites to facilitate PCR amplification and directional subcloning of the T region. This amplicon was subsequently digested with EcoRl & HmdIII and ligated to the pProEx-gstB expression vector (Burns et ah, 2005) which had also been digested with EcoRl & HmdIII. The expression vector containing Υ-gstB was then transformed into E. coli DΗ5α as follows. Competent E. coli cells were removed from -70 ⁰C and allowed to thaw on ice for 30 min. Using ice cold tips, vector (10 μl) was added to cells (200 μl). Samples were stirred gently to mix, and were left on ice for 30 min.

Following this, the samples were heated to 42 ⁰C for 30 sec and placed on ice for 2 min. LB broth (800 μl) was added, and the samples were placed at 37 ⁰C, shaking at 200 rpm for 1 hr. Samples (50 μl) were spread on LB agar plates with 50 μg/ml ampicillin using a glass spreader under sterile conditions. The remaining sample was concentrated by centrifuging briefly and re-suspending the pellets in LB broth (50 μl). This was spread on a LB agar plate with 50 μg/ml ampicillin. The plates were incubated at 37⁰C overnight, and any resultant colonies analysed. Protein expression was induced by the addition of IPTG (0.6 mM final concentration) and monitored by SDS-PAGE and Western blot analysis (Figure 7). Purification of the T-GstB fusion protein was carried out as follows. For T- GstB isolation, cells were lysed in the presence of protease inhibitors

(phenylmethylsulfonyl fluoride (0.1 mM), pepstatin (2 μg/ml) and leupeptin (2 μg/ml)) by the addition of phosphate-buffered saline-sodium deoxycholate (0.5%(w/v)), subjected to DNAse (Sigma, Poole, UK) treatment (final concentration: 10 μg/ml) and the insoluble pellet washed extensively to remove contaminating proteins. Insoluble T-GstB was resuspended in Urea (0.5-1.0 ml, 8 M), containing dithiothreitol (5 mM), at a concentration of 13 mg/ml (Figure 7). Microwells were individually coated with the T-GstB and GstB, respectively (5 μg/ml) for subsequent use in the solid phase 4'phosphopantetheinylation assays. Micro we 11 coating of T-GstB / GstB and subsequent 4'phosphopantetheinylation assays were carried out as described in Section 2.8 for Pes3Aτ- The results are discussed in Section 3.4 below and are shown in Figure 8.

Description of Figures:

Figure 1. Diagrammatic representation of A. Pes3 domain architecture, and B. Thiolation (T)-GstB fusion protein. The first adenylation domain and thiolation domain from module 1 of Pes3 (Pes3Aτ, 1-8 kb) were selected for recombinant protein production. The T-GstB fusion protein is also illustrated showing the Pes3 Thiolation domain (T) fused to the GstB protein. Figure 2. Purification of recombinant PCS3_AT by differential extraction.

A. Coomassie stained SDS-PAGE gel of Pes3Aτ fractions obtained from crude extraction method. Lane M: Molecular mass marker, Lane 1 : unpurified Pes3Aτ , Lane 2: Cell lysate; Lanes 3 - 5: Triton X-IOO washes, Lane 6: 2M urea wash, Lane 7: 8M urea wash, Lane 8: purified Pes3Aτ in 6M Guanidine-HCl.

B. Western blot analysis of Pes3Aτ- Lane M: Molecular mass marker, Lane 1 : unpurified Pes3Aτ, Lane 2: Pes3Aτ purified by differential extraction method. Western blot analysis was carried out using 1/1000 dilution of anti-Hisβ monoclonal antibody and 1/1000 dilution of anti-mouse IgG-HRP conjugate.

Figure 3. Purification of intracellular 4'PPTase from Sf₉ cell lysate. A. SDS-PAGE and B.

Western blot analysis of 4'PPTase purification using anti-Hisβ monoclonal antibody and 1/1000 dilution of anti-mouse IgG-HRP conjugate. Lanes 1 : Molecular mass markers; Lane 2: Control 4'PPTase cell lysate; Lane 3: 4'PPTase cell lysate 1; Lane 4-6: Unbound protein; Lane 7: NiNTA affinity column eluate containing purified 4'PPTase.

Figure 4a. Western blot showing 4'phosphopantetheinylation of Pes3Aτ using a range of 4'PPTase-containing Sf9 cell lysates (20 μl). Final concentrations of Pes3_Aτ (4.44 μM) and biotin-CoA (8.4 μM) were used. Western blots were probed with Streptavidin-HRP

(1/500) and developed with ECL (Pierce Biotechnology).

Lane 1 : 1.7 mg/ml, total cell protein.

Lane 2: 0.85 mg/ml, total cell protein. Lane 3: 0.34 mg/ml, total cell protein.

Lane 4: 0.17 mg/ml, total cell protein.

Lane 5: 0.034 mg/ml, total cell protein.

Lane 6: 0.017 mg/ml, total cell protein.

Lane 7: 0.003 mg/ml, total cell protein. Lane 8: Pes3Aτ negative control.

Lane 9: Cell lysate negative control.

Results shown are representative of duplicate analysis. Figure 4b. SDS-PAGE analysis of 4'phosphopantetheinylation of PCS3_AT by 4'PPTase using Fl-CoA.

(i). Fluorescent analysis was carried out on the Typhoon scanner (Amersham) using 488 nm/520 nm excitation and emission. Lane M: Protein Marker.

Lane 1 & 2: 4'PPTase-containing Sf₉ cell lysate (0.034 mg/ml). Lane 3 & 4: Purified 4'PPTase (0.08 μM). Lane 5 & 6: 4'PPTase negative control. Lane 7: Fl-CoA negative control (Fl-CoA absent). Lane 8: Pes3Aτ negative control (Pes3Aτ absent).

(ii). Coomassie staining of the above SDS-PAGE gel showing identical Pes3Aτ loading (Lanes 1-7).

Figure 5. Solid phase 4'phosphopantetheinylation assay using Fl-CoA. 4'phosphopantetheinylation reactions (including appropriate controls) were coated on black microtitre plates and subsequently analysed by fluorescence with excitation and emission at 490/520 nm, respectively. Results given represent the mean (+/- SE) of the experiment performed in triplicate.

All Reaction constituents: Pes3_Aτ; 4'PPTase; Fl- CoA 4'PPTase Negative Control: 4'PPTase absent from reactions

Ppant Buffer Control: Ppant buffer only

Ppant Buffer and Fl- CoA only: Pes3Aτ and 4'PPTase absent from reactions

Figure 6a. Diagrammatic representation of Solid phase 4'phosphopantetheinylation Immobilised thiolation domains (e.g., in Pes3Aτ) (green circles) are stabilised by the addition of BSA (dashed line). The reactive serine residue (-OH) on the thiolation domain can then be modified with biotin- CoA in the presence of 4'PPTase. 4'PPTase activity is then indirectly detected by addition of streptavidin-horseradish peroxidase (S-HRP) conjugate and tetramethylbenzidine TMB) substrate. Fluorescein-CoA can be substituted for biotin-CoA followed by (subsequent direct fluorescent detection of the labelled thiolation domain.

Figure 6b. Solid phase 4'phosphopantetheinylation assay using Biotin CoA. In vitro 4'phosphopantetheinylation assays were carried out on plates coated with Pes3Aτ (2 μg/ml) using final concentrations of biotin-CoA (5.4 μM), 4'PPTase-containing Sf₉ cell lysate (protein cone: 0.51 mg/ml), and Sf9 cell lysate only (protein cone: 0.51 mg/ml). Results given represent the mean (+/- SE) of the experiment performed in triplicate.

Figure 6c. Solid phase 4'phosphopantetheinylation assay using Fl-CoA. 4'phosphopantetheinylation assays were performed in micro we 11s coated with Pes3Aτ (2 μg/ml) using Fl-CoA (3.6 μM), 4'PPTase-containing Sfg cell lysate (0.51 mg/ml) and Sfg cell lysate only (protein cone,: 0.51 mg/ml). Results given represent the mean (+/- SE) of the experiment performed in triplicate.

Figure 6d. Solid phase 4'phosphopantetheinylation of PCS3_AT using purified PPTase and 4'PPTase cell lysate.

Absorbance values obtained for samples containing control insect cell lysate were subtracted from those obtained with 4 'PPTase cell lysate to give the actual amount of post- translational modification. Results given represent the mean (+/- SE) of the experiment carried out in triplicate. Students t-test was used to calculate P-values for the difference between the purified 4'PPTase and the 4'PPTase lysate (P = 0.56) and biotin-CoA only negative control (P=O.00036). Here, 4'PPTase present in cell lysate (84 μg total protein/reaction) catalyses equivalent post-translational modification of Pes3Aτ as 23 μg purified PPTase.

Figure 7. Expression and purification of recombinant T-GstB.

A. Coomassie stained SDS-PAGE analysis of T-GstB fractions obtained from crude extraction method. Lane M: Molecular mass marker, Lane 1 : unpurified T-GstB lysate,

Lane 2: wash fraction, Lane 3: 8 M urea wash (20 μg), Lane 4: 8M urea wash (2 μg).

B. Western blot analysis of purified T-GstB. Lane M: Molecular mass marker, Lane 1 : T- GstB lysate, Lane 2: Wash fraction, Lane 3 & 4: 8M urea wash (0.5 & 1 μg/track, respectively) T-GstB .

Figure 8. Solid phase 4'phosphopantetheinylation of T-GstB. Absorbance values obtained from samples containing control insect cell lysate were subtracted from those obtained from samples incubated with 4'PPTase cell lysate to give the net amount of post-translational modification. Results given represent the mean (+/- SE) of three individual experiments. Students t-test was used to calculate P-values for the difference between the T-GstB and the GstB (P = 0.0003).

Figure 9a. The effect of temperature on 4'PPTase activity in the presence and absence of protease inhibitors.

Absorbances for insect cell lysate only (protein cone,: 0.51 mg/ml) were subtracted from those obtained with 4'PPTase-containing cell lysates to give the actual amount of post- translational modification at each temperature for both experiments (+/- protease inhibitors). These results were subsequently expressed as % relative to the highest absorbance value, i.e. 24°C with protease inhibitors. Results given represent the mean (+/- SE) of three individual experiments. Students t-test was used to calculate P-values for the difference between the control (24 ⁰C + protease inhibitors) and the following conditions; 4 ⁰C (+ / - Protease inhibitors (P = 0.003; P= 0.0009)), 24 ⁰C ( - Protease inhibitors (P = 0.03)), 37 ⁰C (+ / - Protease inhibitors (P = 0.005; P= 0.0002)) and 50 ⁰C (+/- Protease inhibitors (P = 0.00001; P= 0.00002)).

Figure 9b. The effect of pH on 4'PPTase activity.

Absorbance values obtained from samples containing insect cell lysates were subtracted from absorbance values obtained from samples incubated with 4'PPTase cell lysate to give the net amount of post-translational modification at each pH. These results were subsequently expressed as % relative to the highest level, i.e. pH 8.0. Results given represent the mean (+/- SE) of three individual experiments. Students t-test was used to calculate P-values for the difference between the control (pH 8.0) and the following pH conditions; 4.0 (P = 0.01), pH 6.5 (P = 0.008) and pH 10.0 (P = 0.0003).

Figures 10a - 10c. Sequence Alignments.

Figure 10a: CLUSTAL W multiple sequence alignment of pes3Al (Adenylation Domain 1. Figure 10b: CLUSTAL W multiple sequence alignment of pes3ElC2 (Epimerase Domain 1 -Condensation Domain 2). Figure 10c: CLUSTAL W multiple sequence alignment of pes3C4E2 (Condensation Domain 4-Epimerase Domain 2).

RESULTS

3.1 Recombinant protein expression, purification and solubilisation. PCS3_AT was found to be highly insoluble under non-denaturing conditions (data not shown) and was solubilised by addition of 6 M Guanidine-HCl containing 1 rnM DTT to a final concentration of 30 mg/ml or 5 mg/g cells. SDS-PAGE analysis and Western blot analysis confirmed the required purity of Pes3Aτ (Figure 2). The 72 kDa Pes3Aτ band observed on the Coomassie stained gel was excised and subjected to MALDI-ToF mass spectrometry. Resultant peptide monoisotopic m/z values, compared to those of an in silico digest of Pes3Aτ, revealed that 9/14 peptides corresponded to the Pes3Aτ protein sequence, as shown in Table 4, which represented 20 % sequence coverage thereby providing conclusive confirmation of Pes3Aτ identity.

m/z (Da) Identified Peptides SEQIDNO.

1656.85 KKSVWTMVAMLAIMK 12

1590.88 KRILDDTEAPLVIVHR 13

1523.84 KGIVVPHRSIATSMR 14

1930.95 RWNDLAGAMERLGVNWAK 15

1288.737 RLLHPEQVPSLR 16

1674.8 RQVGGTLHVVDAGNHDR 17

1273.61 HRDEDGVLYHL LGR 18

1531.66 KVAEHSTDTSADNER 19

Table 4. Peptides identified following trypsin digestion of recombinant Pes3_ATand MALDI-ToF mass spectrometry analysis. The peptides identified represent 20 % coverage of the Pes3_AT and confirm the identity of the protein.

Pes3Aτ was solubilised under non-denaturing conditions by dilution into Ppant buffer containing 8M urea (4 ml) at 0.5 mg/ml Pes3Aτ followed by serial dialysis into Ppant buffer (final concentration: 0.41 mg/ml (5.75 μM)). This solubilised Pes3Aτ preparation was aliquotted and stored at -20⁰C for subsequent use.

Following infection with recombinant baculovirus encoding A. fumigatus 4'PPTase, Spodoptera frugiperda insect cell lysates were prepared by sonication in the presence of 0.05%(w/v) sodium deoxycholate in Ppant diluent. Purified 4'PPTase was obtained by incubation with NiNTA affinity resin for 2 hr followed by elution with 250 mM imidazole and dialysis into Ppant diluent (yield: 0.33 mg/10⁸ cells) (Figure 3).

3.2 4'phosphopantetheinylation of Pes3Aτ with biotin- and fluorescent-CoA is mediated by 4'PPTase.

Pes3Aτ was subjected to specific 4'phosphopantetheinylation by 4'PPTase present in insect cell lysates (Figure 4a). Here, modification of the Pes3Aτ (4.4 μM) was clearly detectable using 4'PPTase-containing the insect cell lysates (17 - 1700 μg/ml total protein), with a weak signal, corresponding to specific 4'phosphopantetheinylation, at 3 μg/ml total cell protein. No 4'phosphopantetheinylation was detectable in the presence of control insect cell lysate thereby providing confirmation that modification of Pes3Aτ with biotin-CoA was mediated specifically by 4'PPTase and not as a result of direct non-specific labelling of Pes3Aτ with residual N-hydroxysuccinamido-biotin. Omission of Pes3Aτ from the reaction likewise produced a negative result which confirmed that proteins in the insect cell lysate were not labelled non-specifically with biotin-CoA (Figure 4a). Replacement of biotin-CoA with fluorescein- CoA as the 4'PPTase substrate also facilitated post- translational modification of Pes3Aτ (Figure 4b). Here, Pes3Aτ (4.44 μM) was labelled using both purified 4'PPTase (0.08 μM) and 4'PPTase present in insect cell lysate (34 μg/ml total cell protein). No 4'phosphopantetheinylation was detectable in the absence of either 4'PPTase or Pes3Aτ- Overall, these observations confirm that the Pes3Aτ thiolation domain can be post-translationally modified by 4'PPTase with either biotinylated or fluorescently labelled CoA.

3.3 High-throughput detection of 4'phosphopantetheinylation.

Reaction mixtures containing combinations of purified 4'PPTase, Pes3Aτ, Fl-CoA were incubated for 1 hr and subsequently transferred to black microtitre plates (1 hr) following by washing using PBST to remove unbound Fl-CoA. Fluorescent analysis at 490/520 nm (ex/em) (Figure 5) indicated that fluorescently labelled Pes3Aτ had bound to the plate and was detected by fluorescence analysis. Fluorescence labelling was predominantly mediated by 4'PPTase modification, however non-specific labelling was also evident whereby fluorescence values approximately 25% those of test samples were observed in the absence of 4'PPTase. No fluorescence was detectable in the absence of Pes3Aτ- 4'Phosphopantetheinylation using biotinylation of immobilised PCS3_AT was investigated using microwells pre-coated with Pes3Aτ (2.8 pmol), and stabilised by BSA addition (Figure 6a). To ensure that biotin labelling of the bound Pes3Aτ protein was the result of 4'PPTase activity and not due to the presence of insect cell proteins present in the crude cell lysate, control reactions were included whereby uninfected insect cell lysates (at identical total protein concentration) were incubated in the Pes3Aτ coated microwells in the presence of biotin-CoA, and resultant absorbance values compared to those obtained from incubation with 4'PPTase insect cell lysate. Higher absorbance values were obtained in this control sample in comparison to the biotin-CoA and Ppant buffer control (A450/630 ■ 0.44 vs 0.28), but the values were significantly lower than the values obtained for the wells incubated with 4'PPTase insect cell lysate (A45o/63θnm 1.7), thereby confirming that labelling of Pes3Aτ was due to 4'PPTase present in the insect cell lysate. Consequently, 4'PPTase cell lysate was used for subsequent analysis of 4'PPTase activity using this assay format (Figure 6b). Control insect cell lysate (uninfected cells) was also included in subsequent analysis, and the absorbance values obtained for these samples subtracted from the absorbance values obtained from 4'PPTase samples to correct for any non-specific labelling that may be a result of native insect cell proteins. 4'PPTase-mediated Fl-CoA modification of immobilised Pes3Aτ has also been demonstrated (Figure 6c). Here, nonspecific modification (~ 25 % of the 4'PPTase reaction) was also evident.

Purified 4'PPTase from insect cells lysates also functions in the solid phase 4' phosphopantetheinylation reaction (Figure 6d). No significant difference was observed between purified 4'PPTase and 4'PPTase cell lysate (P = 0.56), whereas significantly greater 4 'phosphopantetheinylation activity was evident compared to biotin-CoA only negative control (P=O.00036). The activity equivalence between purified 4'PPTase and the 4'PPTase cell lysate (Figure 6d) suggests that the enzyme comprises approximately 30% of total cell protein.

3.4 Fungal thiolation domain facilitates labelling of non-native substrate proteins. The 36 kDa T-GstB fusion protein was purified from E.coli cell lysates resulting in a T- GstB yield of 13 mg/ml or 5 mg/g cells (Figure 7). T-GstB was post-translationally modified by 4' phosphopantetheinylation with a biotin tagged Ppant residue when immobilised in microwells (Figure 8). Moreover, background signal arising from GstB negative control was less than 5% of overall T-GstB signal, demonstrating conclusively that the discrete thiolation domain of T-GstB was specifically modified by 4'PPTase activity.

3.5 Characterisation of 4'PPTase activity. Following the establishment of the in vitro 4'phosphopantetheinylation assay in a solid phase (96 well plate) format using biotin-CoA and 4'PPTase insect cell lysates, temperature and pH optima, as well as reaction time were investigated. Analysis of this data revealed that optimal 4'PPTase activity was evident at 24 ⁰C, with reduced 4'phosphopantetheinylation observed at 4°C, 37°C and 50⁰C (Figure 9a). In the presence of protease inhibitors, proteases present in the crude insect cell lysate might be degrading 4'PPTase and therefore lowering the extent of post-translational modification occurring. Data from this experiment can be seen in Figure 9a. The overall affect of pH on 4'PPTase activity is shown in Figure 9b whereby background absorbance values obtained from the insect cell lysate control samples were subtracted from the absorbance values obtained from 4'PPTase lysate reactions. From this analysis it is clear that pH has an effect on the activity of 4'PPTase with optimal 4'phosphopantetheinylation occurring at pH 8.0.

3.6 Sequence Alignments

A number of regions (Pes3Al, Pes3ElC2 and Pes3C4E2) from the pes3 gene from A. fumigatus strain 26933 were cloned and sequenced to ascertain (i) if the full length gene was present in strain 26933 and also (ii) to identify regions which could be subjected to expression analysis by RT-PCR to evaluate pes3 gene expression in A. fumigatus 26933.

The identification of putative NRP synthetase pes 3 gene was facilitated by the availability of the recently completed A. fumigatus genome (A. fumigatus ATCC293) at The Institute of Genomic Research (TIGR) (www.tigr.org) and also the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih. gov), and through the Central Aspergillus Data Repository (CADRE) (www.cadre-genomes.org.uk). A bioinformatic approach which involved data mining of the genome using the previously identified NRP synthetase, enniatin synthetase (NCBI accession: Zl 8755), was initially employed to search for putative regions of homology. This interrogation of the A. fumigatus genome resulted in the identification of an open reading frame (ORF) encoding a putative NRPS of ~ 25 kb in size. This nucleotide sequence was translated to an amino acid sequence using the translation tool at vv^vw,_.cxpasχorg, giving rise to a protein containing 8515 amino acids and therefore predicted to encode a significantly large protein of- 940 kDa.

Bioinformatic analysis of the data obtained from the sequencing of the A. fumigatus genome provides a means of identifying putative NRPSs within this fungus. This can be carried out using a basic local alignment search tool (BLAST) at http://www.ncbi.nlm.nih. gov/cgi-bin/ BLAST/ which takes a query sequence and searches for sequences with regions of homology within a database. The results generated from this Blast search are comprised of sequences with associated bit scores and E values where a high bit score reflects that the two sequences are closely related and conversely, a small E value shows that it is less likely that the similarity occurred by chance and therefore reflects common ancestry. NRPSs are made up of distinct catalytic units or domains and include adenylation, thiolation and condensation domains. The individual domains and modular organisations of the identified NRP synthetase sequences can then be elucidated by carrying out a conserved domain search using BLAST or using the pfam (protein families database of alignments) tool. Having identified putative NRP synthetase sequences, a multiple sequence alignment using either the ClustalX or ClustalW programme can be carried out which compares DNA or amino acid sequences and aligns them to determine regions of homology or to identify conserved motifs within a sequence.

Isolation of Genomic DNA from A. fumigatus - Small Scale.

Aliquots of culture media (10 ml) were inoculated with A. fumigatus conidia and incubated at 37 ⁰C for 24 - 48 hr with constant agitation at 200 rpm. Mycelia were harvested from the cultures and placed in 2 ml microfuge tubes with extraction buffer (400 μl) and the mycelia suspensions sonicated (Bandelin Sonopuls, Progen Scientific Ltd, UK) for 3 x 5 s at a maximum power. Phenol:chloroform:isoamyl alcohol (25: 24: 1) (400 μl) was added to the resultant lysates and the tubes shaken vigorously until the contents were fully emulsified, followed by centrifugation at 13,000 x g for 5 min to separate the phases. The upper aqueous phase (~ 500 μl) was removed to a fresh 2 ml tube and the DNA precipitated by addition of 0.1 x volume of 3 M NaOAc pH 5.2 and 2 x volumes of 100 % (v/v) EtOH, followed by incubation at -20 ⁰C for 30 min. DNA was pelleted by centrifugation at 13,000 x g for 10 min, and the pellets washed with 70 % (v/v) EtOH. After air-drying briefly, DNA pellets were resuspended in 50 μl TE buffer. Polymerase Chain Reaction Amplification of pes3 Regions in genomic DNA.

Polymerase chain reaction (PCR) was predominantly carried out using AccuTaq LA polymerase (Sigma) with the exception of using the ReddyMix system (ABgene) for large scale screening of fungal transformants. This comprised of reaction buffer, Taq polymerase, dNTPs and agarose gel running buffer in a 2 x mixture which was stable at 4 ⁰C. The general reaction constituents for both polymerases used was as follows:

AccuTaq LA polymerase

1OX reaction buffer 2 μl dNTP mix (lO μM) 2 μl

Primer 1 (100 pmol/μl) 1 μl

Primer 2 (100 pmo 1/μl) 1 μl

DMSO 0.8 μl

DNA template 1-10 ng Sterile water to a total of 20 μl

Ab gene Readymix System

2 x PCR mixture 10 μl

Primer 1 (100 pmol.μl) 1 μl Primer 2 (100 pmo l.μl) 1 μl

DNA template 1-10 ng

Sterile water to a total of 20 μl

The following reaction cycle was used unless otherwise stated:

95 ⁰C (denaturing) 1 min

95 ⁰C (denaturing) 1 minPl

55 ⁰C (annealing) 1 min [ x 30-40 cycles

72 ⁰C (extending) 1 min 72 ⁰C (extending) 5 min

Annealing temperatures were estimated as ca. 1O⁰C below the melting temperature (T_m) of the primers used. Extension times used were ca. 1 min/kb of DNA to be synthesised. Reactions were carried out using a PCR Express cycler (Hybaid, MA, USA) or a Bio-Rad Gene Cycler.

DNA Sequencing DNA sequencing of recombinant clones was performed by Lark Technologies (Essex, U.K.), and Westburg sequencing (The Netherlands), on a commercial basis.

PCR Amplification of pes3

In order to confirm the presence of pes3 in the A. fumigatus ATCC293 genome, PCR amplification of specific domains within the ORF was carried out. Primers were designed to amplify a 1.2 kb fragment predicted to encode the first Adenylation domain in the synthetase (pes J-Al) and two 1 kb regions spanning across a predicted condensation domain and epimerisation domain (pes3-ΕlC2, pes3-Ε2C4).

Primers were designed with restriction sites suitable for subsequent cloning into the TOPO TA Cloning vector and are listed in Table 5. A. fumigatus ATCC26933 genomic DNA was isolated from mycelia harvested from a 72 hr culture grown in 5 %(v/v) foetal calf serum in minimal essential media and used as a template for PCR. AccuTaq polymerase was employed for amplifying all regions of pes 3. PCR products obtained were electrophoresed on a 1 % (w/v) DNA agarose gel and visualised using ethidium bromide staining and a Stratagene Eagle Eye II digital still video system.

Primer (F/R) Sequence (5'-3')

^"PesΪAT Forward GAGACTCGAGATGTCCAACAACACAATG (SEQ ID NO. 26) 5

Pes3Al Reverse GAGAAAGCTTGCATCAGTCCGAGATTGATCC (SEQ ID NO. 27)

Pes3ElC2 Forward GAGACTCGAGCCTAACATTGTTCCC (SEQ ID NO. 28)

Pes3ElC2 Reverse GAGAAAGCTTATCCTCATCCAGGCTGGC 10 (SEQ ID NO. 29)

Pes3E2C4 Forward GAGATCTAGATTGGCCAAGGATGTTTCC (SEQ ID NO. 30)

Pes3E2C4 Reverse GAGAGAATTCGCGAGCCAAAATTGC (SEQ ID NO. 31) 15

Table 5. Oligonucleotide primers designed for amplification of pes3 fragments.

TOPO TA Cloning of pes3 Fragments and Subsequent Sequence Analysis Having obtained PCR products for each of the required pes 3 regions, it was necessary to prepare stocks of these regions by subcloning into a PCR^® 2.1 TOPO cloning vector (Invitrogen). Trans formants were screened for the presence of the specific insert by PCR amplification with the appropriate primers. A PCR positive result was obtained for each insert confirming the successful cloning of pes 3 Al, pes 3ΕIC2 andpes3Ε2C4 into the TOPO vector. In order to obtain as much information as possible on the pes3 gene sequence and to investigate differences between the sequenced strain 29633 and A. fumigαtus ATCC293, plasmid DNA was isolated from the positive transformants and subjected to sequence analysis using the M 13 forward and reverse primers supplied with the TOPO cloning vector which amplify sequences within the multiple cloning site. The resultant sequence data was aligned and compared to the theoretical /?es 3 data and showed overall similarity of greater than 99 % with 7 base differences in total. The sequence data from the various pes 3 fragments was subsequently translated and aligned to the corresponding theoretical protein sequence of Pes3 in order to see the actual amino acid differences throughout the sequences. Observed differences were minimal with 2 amino acid differences observed. The sequence alignments of pes 3 -Al, pes 3 -Ε1C2 and pes 3- E2C4 are shown in Figures 10a - 10c respectively. Examination of the pes 3- Al sequence alignment identified 1 amino acid difference when compared to the theoretical data. This difference revealed a conservative change however from M to V at position 138. The pes3- C2E1 sequence alignment revealed 1 conserved amino acid change from E to Q at position 54. Analysis of the remaining pes3-Ε2C4 alignment revealed 100 % sequence identity. All PCR fragments were cloned and sequenced twice and resulted in the same amino acid changes, indicating true polymorphism rather than a sequence error.

COMPARATIVE EXAMPLES

MATERIALS AND METHODS

4.1 For comparison with the invention which involves immobilisation of the PCP domain (immobilisation of the PCP part of Pes3Aτ), attempts were made to add solution phase assay constituents (e.g. biotin-CoA, 4'PPTase, PCP (Pes3Aτ)) which had been allowed to react and therefore contained enzymatically-modified biotinylated PCP, to microtitre plates, for subsequent detection by streptavidin-horseradish peroxidase. The reaction constituents were as follows: 1. All Reaction constituents: Pes3_Aτ; 4'PPTase; Biotin CoA 2. 4'PPTase Negative Control: 4'PPTase absent from reaction

3. Ppant Buffer Control: Ppant buffer only

4. Ppant Buffer and biotin-CoA only: Pes3Aτ and 4'PPTase absent from reaction.

The reaction constituents were prepared according to the method described in Section 2.7, using Pes3_Aτ (4.4 μM), 4'PPTase (0.08 μM), biotin-CoA (8.4 μM) in 75 mM Tris-HCl, 5 mM DTT and 10 mM MgCl₂, pH 8.0 (final volume: 100 μl). The reaction constituents were added into 1.5 ml tubes and incubated for 1 hr at 24°C for phosphopantetheinylation to occur, and subsequently transferred into wells of maxisorb 96 well plates. This assay was carried out in triplicate and included suitable controls, (i.e. Ppant buffer only, Ppant buffer and biotin-CoA only and reaction constituents without 4'PPTase. The results are shown in Figure 11.

4.2 In order to determine whether it was the biotin or the CoA binding to the plate, the assay described in 4.1 above (and according to Section 2.7) was repeated, with the addition of a 10 fold molar excess of exogenous CoA (2 μM) into the reaction mixture after the 1 hr incubation at 24°C. The results are shown in Figure 12.

4.3 In an attempt to overcome the problem of biotin-CoA directly binding to the plate, an alternative method was employed to develop the biotin-CoA based in vitro

4'phosphopantetheinylation assay to a microtitre plate format. This alternative method was according to the invention and involved pre-coating microtitre plates, as per Section 2.8, with Pes3Aτ prior to the addition of 4'PPTase and biotin-CoA (Figure 6a). The results are shown in Figure 13.

Description of Figures:

Figure 11. Solid phase in vitro 4'phosphopantetheinylation assay using previously mixed reaction mixtures. After reaction, the in vitro 4'phosphopantetheinylation reaction mixtures (including appropriate controls) were coated on microtitre plates and probed with streptavidin-HRP prior to addition of substrate TMB. Absorbance was determined at 450/630nm. It is clear that streptavidin-HRP binding, and subsequent colour development occurs (i) even in the absence of 4'PPTase and (ii) when biotin-CoA only is present in microwells.

Figure 12. Investigation of the binding capacity of biotin-CoA to microtitre plates by addition of excess CoA.

CoA (2 μM) was added to the reaction tubes post incubation at room temperature and the resultant samples were subsequently applied to the microwells for coating to occur as per Figure 11. A control comparative experiment was carried out in parallel without the addition of CoA.

1. All Reaction constituents: Pes3Aτ; 4'PPTase; biotin-CoA

2. 4'PPTase Negative Control: 4'PPTase absent from reactions

3. Ppant Buffer Control: Ppant buffer only 4. Ppant Buffer and biotin-CoA: Pes3Aτ and 4'PPTase absent from reactions

Figure 13. Investigation of the effect of the invention, i.e. pre-coating plates with PCP domain (referred to in Figure 13 as Pes3-Modl - another name for Pes3Aτ) on the ability of biotin-CoA to bind to the microwells of the 96 well plates. Microwells coated with PCS3_AT (2 μg/ml) were incubated with biotin-CoA (5.4 μM) diluted in Ppant buffer, and with Ppant buffer only. A control experiment was carried out in parallel whereby uncoated plates were used for comparative analysis. It is clear that PCP domain presence on microwells prevents non-specific binding of biotin-CoA.

RESULTS

4.4 Prior immobilisation of PCP domain is essential for assay function: Referring to Figure 11, the results revealed that negative control samples containing Ppant buffer and biotin-CoA only, gave rise to the highest absorbance values, indicating that the biotin-CoA was binding directly to the plate and therefore rendering the this method an unsuitable format for detecting 4'PPTase activity. Coating of reaction mixtures on microplates was unsuccessful because binding of biotin moiety of biotin-CoA to microtitre plates was observed. This confirms that the method of the invention comprising immobilisation of PCP to the microtitre plates is absolutely essential to obtaining a functional phosphopantetheinylation assay. Furthermore, the fact that the 4'PPTase Negative Control (wherein 4'PPTase which is essential to the reaction is absent) produced a similar absorbance (i.e. a false positive) to the absorbance obtained using all reaction constituents is further indication of the undesirable binding of biotin-CoA directly to the plate. These results can also be compared with the results shown in Figures 6b and 6c and discussed in Section 3.3, relating to the invention and in which, when a control PPTase is used, the absorbance is significantly reduced compared to using all of the reaction constituents, showing that the invention is working.

Referring to Figure 12, the addition of a 10 fold molar excess of exogenous CoA (2 μM) into the reaction mixture after the 1 hour incubation at 24°C did not inhibit non-specific binding and confirms that the biotin moiety (as opposed to the CoA moiety) binds non- specificially to microplates.

With reference to Figure 13, in order to test whether coating plates with Pes3Aτ in accordance with the invention prevented the direct, non-specific binding of biotin-CoA to the microwells observed in Figure 11 and described above in Section 4.1, experiments were carried out with the exception that biotin-CoA and Ppant buffer were only applied and the reaction samples were incubated in the Pes3Aτ pre-coated wells. A control experiment was carried out in parallel using uncoated plates. The results from this experiment show that pre-coating plates with Pes3Aτ decreased the non-specific binding capacity of biotin-CoA to the microwells of the 96 well plates by 80 %.

Therefore, not only does the immobilised PCP domain act as an acceptor substrate for biotinylated phosphopantetheine but it actually prevents non-specific, and unwanted, biotin-CoA binding to the microwells. This is absolutely central to correct functioning of the present invention. It has now surprisingly been found that the reaction is not possible by simply adding the reaction constituents into the well, the attachment of PCP to the well is necessary, and the attachment of biotin-CoA to the well is clearly undesirable. In summary, it has surprisingly been found that by immobilising the carrier protein domain on the support, it is possible to provide a method for investigating protein phosphopantetheinylation and detecting 4'PPTase activity which is simple, fast, high- throughput and cost-efficient.

Claims

CLAIMS:

1. A method for detecting 4'-phosphopantetheinyl transferase (4'PPTase) activity, the method comprising the steps of:

(a) immobilising a carrier protein domain or a functional fragment thereof on a solid support suitable for this purpose;

(b) reacting the immobilised carrier protein domain or functional fragment thereof of step

(a) with labelled coenzyme A and 4'PPTase to form a labelled carrier protein or functional fragment thereof; and

2. A method for detecting 4'-phosphopantetheinyl transferase (4'PPTase) activity, the method comprising the steps of:

3. A method as claimed in claim 1 or claim 2, wherein the carrier protein domain or functional fragment thereof has a non-native protein fused thereto.

4. A method for the indirect labelling of a non-native protein, the method comprising the steps of: (a) providing a fusion protein comprising a carrier protein domain or a functional fragment thereof and a non-native protein fused thereto;

(b) immobilising the fusion protein on a solid support suitable for this purpose via the carrier protein domain or functional fragment thereof; (c) reacting the immobilised fusion protein of step (b) with labelled coenzyme A and 4'PPTase to form a labelled carrier protein domain or functional fragment thereof, thereby indirectly labelling the non-native protein; and optionally

5. The invention still further provides a method for the indirect labelling of a non- native protein, the method comprising the steps of:

(a)(i) providing a fusion protein comprising a carrier protein domain or a functional fragment thereof and a non-native protein fused thereto;

6. A method as claimed in any one of claims 3 to 5, wherein the non-native protein is selected from the group comprising antibodies, antigens, enzymes and receptors, wherein, preferably, the protein is an enzyme, more preferably a glutathione ^-transferase (GST).

7. A method as claimed in any preceding claim, wherein the carrier protein domain or functional fragment thereof is an acyl carrier protein (ACP) domain or a peptidyl carrier protein (PCP) domain, preferably a PCP domain.

8. A method as claimed in any preceding claim, wherein the carrier protein domain or functional fragment thereof is derived from or is a component of an enzyme selected from a nonribosomal peptide (NRP) synthetase, a fatty acid synthetase and a polyketide synthetase, preferably an NRP synthetase.

9. A method as claimed in claim 8, wherein the enzyme is derived from a fungus or a bacterium wherein, preferably, the fungus is selected from the group comprising Aspergillus fumigatus, Aspergillus nidulans, Cochliobolus heterostrophus, Trichoderma virens, Magnaporthe grisea, Trichoderma harzianum, Claviceps purpurea, Gibberella zeae, Fusarium graminearum and Penicillium spp., more preferably Aspergillus fumigatus; and wherein, preferably, the bacterium is selected from the group comprising Pseudomonas spp., Angiococcus disciformis, Mycobacterium tuberculosis, Myxococcus xanthus, Xanthomonas albilineans, Streptomyces spp, and cyanobacteria.

10. A method as claimed in any preceding claim, wherein the 4'PPTase is obtained from host cells selected from bacterial cells, yeast cells, mammalian cells, plant cells and insect cells, preferably recombinant insect cells, more preferably recombinant Spodoptera frugiperda_? (Sfc>) insect cells.

11. A method as claimed in any preceding claim, wherein the coenzyme A is labelled with biotin, fluorescein, a protein, a single- or double-stranded nucleic acid, or a carbohydrate, wherein, preferably, the coenzyme A is labelled with biotin.

12. A method as claimed in any preceding claim, wherein the solid support comprises a plastics material, glass or metal.

13. A method as claimed in any preceding claim, wherein the step of detecting the presence of the labelled carrier protein domain or functional fragment thereof comprises measuring the amount of labelled carrier protein domain or functional fragment thereof formed.

14. A kit for use in the method of any preceding claim, comprising:

(a) a solid support;

(c) labelled coenzyme A; and (d) 4'PPTase; wherein the components listed in (a) - (d) are as defined in any preceding claim.

15. A kit as claimed in claim 14, wherein the carrier protein or functional fragment thereof comprises a non-native protein fused thereto.