CN104136607A - Biologically active proteins having hotspots for directed crosslinking and immobilization - Google Patents

Abstract

The present invention relates to a novel, biologically active loop mutant protein with sustained stability relative to the native protein having hotspots located away from the active center for directed immobilization and crosslinking properties. The invention further relates to a method of genetically engineering of said biologically active proteins, as well as further biological materials modified in accordance with the present invention.

Description

There is the biological activity protein of and immobilized focus crosslinked for orientation

Technical field

The present invention relates to have the novel bioactive ring mutain of lasting stability compared with native protein.Another aspect of the present invention is to insert the method for genetically engineered biological activity protein, particularly enzyme via retaining ring.

More specifically, the present invention relates to have lasting stability and active novel ring mutain, particularly enzyme compared with native protein, its have be positioned at active centre at a distance for the oriented immobilization of albumen and crosslinked focus.Another aspect of this specific invention is to insert the method for genetically engineered these albumen, preferred enzyme via retaining ring, and described method is for setting up for oriented immobilization and crosslinked focus.

Background technology

Enzyme is biological catalyst, and due to their high reactivity, selectivity and specificity, they are widely used in commercial run.In all enzymes, it is very important material that cellulase (enzyme of degraded cellulose) is becoming for biotechnology and enzyme engineering.This is because Mierocrystalline cellulose is polymkeric substance the abundantest on the earth.Cellulase is being used to multiple industrial object more and more, therefore, will make great efforts to be in a large number put to their expression and the research of their site-directed mutagenesis.

In nearest 30 years, the application of cellulase in commercial run has been increased to sizable amount.Cellulase is widely used in industrial textile, to improve fabric quality, wash (biostoning) to obtain popular aging outward appearance for the enzyme of denim goods clothes especially for the biological fine processing (biopolishing) of textiles.They are also widely used in feed, foodstuffs industry, are used in paper pulp and paper conversion, and are used in laundry.In nearest 10 years, several research focuses on and transforms lignocellulose biomass to produce the substituting renewable energy source of biofuel as fossil oil with cellulase.

Much research proposes to strengthen with linking agent the stability of enzyme, but they seem that activity is had to adverse influence, because their targets also may be present near the amino acid of some type of active centre.The reagent such as known and glutaraldehyde crosslinked can by the Conformational flexibility of restriction enzyme (due to the non-specific binding of lysine residue) reduce enzymic activity (people such as Busto, 1997; The people such as Domen, 1990).

Conventionally be knownly immobilized on upholder or crosslinked enzyme and other biomolecules has the performance as the improvement of biosensor or biological catalyst.In addition, they can be reclaimed and are used for reusing.But, use available techniques to depend on the performance of biomolecules and the performance on immobilization support or surface to the immobilization height of biomolecules.In most of the cases, immobilization or crosslinked can cause the decline of enzymic activity or the ligand binding performance of interfere with biomolecules.Great majority can be used for being cross-linked or immobilized method can not realize directed combination.This event is an almost random process, wherein can be from much different loci fixed target albumen, and this can cause the decline of enzymic activity.For example, much research proposes to strengthen with linking agent the stability of enzyme, but they seem that activity is had to adverse influence, because their targets also may be present in amino acid (people such as Busto, 1997 of near some type active centre; The people such as Rao, 1998; The people such as Yuan, 1999).

Oriented immobilization is the current method reducing for overcoming this biological activity.Change for immobilization or crosslinked albumen orientation is problematic, and normally case is specific.In many cases, this idea is used to promote transfer transport people such as (, 2002) Ferapontova of the enzyme from upholder to redox biosensor.For this purpose, use the cysteine residues on target enzyme that described enzyme is fixed on the upholder that contains disulphide group or be made of gold.But there is a significant drawback in the method.It is not the cysteine residues that all biomolecules all contain the oriented immobilization of free cysteine residues or its position permission biomolecules.Application No. 2009/0120809 has been described a kind of nitroreductase, and it is modified to and comprises multiple cysteine residues that mix in its structure, for by described enzyme oriented immobilization at noble metal electrode.In addition, some research has solved this problem (people such as Ferapontova, 2002 by cysteine residues is introduced in enzyme surface; The people such as Gwenin, 2007; The people such as Kallwass, 1993; The people such as Kapp, 2006; The people such as Viswanath, 1998).But the application of the method is limited to not always favourable disulphide-mercaptan exchanges chemistry.

In addition, in some cases, directly change albumen by site-directed mutagenesis technology and be orientated for immobilization.Some situation relates to application (people such as Nakamura, 2011 of histidine residues or the His label of location exchange; The people such as Porath, 1975).Conventionally these are fixed on (people such as Andreescu, 2001) on immobilized metallo-chelate (IMAC) or golden upholder.But a significant drawbacks of the method is that, after adding emulative part, immobilization is reversible (Jerker, 1992).In addition, often His-label is added into N-or the C-end of target protein, therefore this can limit the suitable orientation that will only be immobilized into the albumen on N-and C-end.This can limit the application of the method in some albumen, and described albumen is correctly directed in meeting after N or the immobilization of C-end.

The enrichment of some reactive group on protein surface is the another kind of method that instructs the oriented immobilization of biomolecules.For this purpose, the people such as Guisan introduce 3 lysine residues (people such as Abian, 2004) on the surface of penicillin G acylase of being rich in Lys group.Immobilization speed on verified, glyoxyl-based-agarose has increased and has exceeded 10 times, and the thermostability of enzyme increases.The people such as Terreni have designed such albumen: it has the label of 3 Methionins that replace with 3 glycine on the C-end end of the β of same enzyme chain, and described label has also improved immobilization on PGA people such as (, 2009) Serra.A kind of rear method has the shortcoming identical with using His label, because the application of the method is limited to some albumen.If active centre is near N or the C-end (insert herein Methionin ring, thereby cause active decline) of albumen, the application also may have shortcoming.

The combination of preceding method is also applicable in document.For example, PCT patent application WO 03/044189 has described modified albumen, be modified HIV-1 capsid protein p24, it is by comprising nucleotide fragments (its coding at least 6 lysine residues continuously) nucleotide sequence coded that is known as poly-K, the albumen modified with the second, it is by comprising nucleotide fragments (its coding at least 6 histidine residues continuously) nucleotide sequence coded that is known as poly-H.These polylysines and polyhistidyl label are also positioned at N-end or the C-end of albumen.Therefore,, as described in the early time, the method has limited application.

In the present invention, insert amino acid ring to be positioned at away from the mode in the active centre of albumen, to set up for crosslinked and immobilized local high-affinity point.

Successfully improve the operational stability of albumen, preferred enzyme.In the present invention, produced modified albumen, it contains for oriented immobilization or crosslinked lip-deep focus.For this purpose, preferably use site-directed mutagenesis that the interior ring (endo-loop) that is rich in Methionin and glycine is inserted in the surface of enzyme, to set up for immobilization and crosslinked local high-affinity point.The ring position of selecting is the height come-at-able and flexible site of solvent, and it is positioned at away from active centre place, so that activity that can interferases.The insertion of interior hoop albumen is problematic process because the sequence of inserting may jamming target synthetic, folding pathway and the overall structure of albumen.In fact,, in those tests of reporting in prior art, confirmed that interior ring mutain is (Amatore and Baneyx, 2003 of functional disorder limbic function or complete; The people such as Doi, 1997; The people such as Doi, 1998; The people such as Heinis, 2006; Manoil and Bailey, 1997).With the surprising contrast of prior art in, find that interior ring mutant enzyme of the present invention is completely functional, its implication is that such mutant has retained all biological activitys compared with wild-type enzyme.In many cases, be also noted that the lasting stability of folded state compared with natural enzyme.

Summary of the invention

In the present invention, comprise and have lasting stability and active Novel ring mutain compared with native protein, it has the focus being positioned at away from active centre place, to the oriented immobilization of albumen is provided and is cross-linked.

Particularly, the present invention relates to novel bioactive mutain, the non-natural structural element that all essential tertiary structure element that it comprises corresponding native protein and at least one surface expose, it is characterized in that, described non-natural structural element is positioned at albumen primary structure and forms the peptide ring that surface exposes, and described peptide ring is set up focus to allow the enhanced stability of directed proteopexy and folding albumen tertiary structure.

The non-natural structural element that all essential tertiary structure element that described bioactive mutain comprises corresponding native protein and at least one surface expose, it is characterized in that, described non-natural structural element is positioned at albumen primary structure and forms the peptide ring that surface exposes, described peptide ring is set up focus to allow the enhanced stability of directed proteopexy and albumen tertiary structure, and wherein said native protein is preferably selected from and comprises enzyme, antibody, acceptor, antibody fragment, albumen in conjunction with albumen and synthetic peptide.

Another preferred embodiment of this concrete invention is above-mentioned novel bioactive mutain, preferably enzyme of wherein said mutain, and preferred cellulose enzyme, more preferably endoglucanase (EGI), the endoglucanase of most preferably extracting from Trichodermareesei (Trichoderma reesei).

Another preferred embodiment of the present invention is bioactive ring mutain, it sets up focus to allow the enhanced stability of directed proteopexy and albumen tertiary structure, there is the non-natural structural element that a surface exposes, it is characterized in that, described non-natural structural element is positioned at albumen primary structure and forms the peptide ring that surface exposes, it is characterized in that, described non-natural structural element be comprise at least 5 and at the most 15 amino acid, most preferably comprise 10 amino acid whose amino acid rings.

Another preferred embodiment of the present invention is bioactive ring mutain, it has the non-natural structural element that a surface exposes, it is characterized in that, described non-natural structural element is positioned at albumen primary structure and forms the peptide ring that surface exposes, this can set up focus to allow the lasting stability of directed proteopexy and albumen tertiary structure, it is characterized in that, described non-natural structural element is the ring that comprises Methionin and glycine aminoacid sequence, and described ring preferably comprises Methionin and glycine aminoacid sequence alternately, most preferably be defined as the sequence of KKGGKKKGGK.

Most preferably, mutant enzyme described herein is the endoglucanase of extracting from Trichodermareesei, and it contains the ring on the almost offside of 180 ° that is positioned at active centre; In the come-at-able region of height solvent, and average at the C α apart from active centre residue the 112nd and the 113rd residue between flexible region in.

Another aspect of the present invention is the method for genetically engineered bioactive mutain, described method is characterised in that following steps: (1) limits site along the exon region of target protein gene, described site is for inserting the oligonucleotide of the ring that forms surface exposure, (2) sequence of restriction DNA primer, described sequence comprises the end overlapped ends that forms the oligonucleotide bridging encircling by described, described primer is designed to the area hybridization along the insertion point of described selection via described overlapped ends, (3) chemical synthesising DNA primer, described DNA primer comprises the described overlapped ends that forms the oligonucleotide sequence bridging encircling by described, (4) apply overlapping PCR elongation technology, contain the mutator gene of the oligonucleotide sequence of described formation ring in the designated area of the exon of target protein gene with preparation, (5) apply suitable DNA isolation technique with mutator gene described in purifying, (6) express described mutator gene, (7) mutant of the described target protein that separation contains described peptide ring.

According to another aspect of the present invention, provide oligonucleotide sequence, it is peptide ring after gene expression dose is specifically designed to form translation.More specifically, oligonucleotide code packages is containing the endo glucanase gene of modifying by adding multiple codons of Methionin and glycine residue by site-directed mutagenesis.Preferably, described gene is selected from the codon optimized form of Trichodermareesei (QM9414) egl1 gene.The nucleotide sequence of codon optimized Trichodermareesei (QM9414) the egl1 gene with modifying provides as SEQ NO 1, and most preferably, described endo glucanase gene is by following sequence encoding: the nucleotide sequence of describing in SEQ NO 1, the reverse complement of described sequence, the complement of described sequence, the reversion of described sequence, or there is the sequence of at least 60% sequence identity with any the nucleotide sequence in aforementioned sequence:

SEQ NO 1：

>egl1_L5_ gene

gaattccagcaaccgggtacctctactccagaggttcacccaaagttgactacttacaagtgtactaagtccggtggttgtgttgctcaagacacttccgttgttttggactggaactacagatggatgcacgacgctaactacaactcctgtactgttaacggtggtgttaacactactttgtgtccagacgaggctacttgtggaaagaactgtttcatcgagggtgttgactatgctgcttccggtgttactacttctggttcctccttgactatgaaccagtacatgccatcttcctctggtggttactcttctgtttccccaagattgtacttgttggacaagaagggtaagaagaagggtaagaaatccgacggtgaatacgttatgttgaagttgaacggtcaagagttgtctttcgacgttgacttgtccgctttgccatgtggtgaaaacggttccttgtacttgtctcagatggacgaaaatggtggtgctaaccagtacaatactgctggtgctaactacggttctggttactgtgatgctcagtgtccagttcagacttggagaaacggtactttgaacacttcccaccagggattctgttgtaacgagatggacatcttggagggaaattccagagctaacgctttgactccacactcttgtactgctactgcttgtgactctgctggttgtggttttaacccatacggttccggttacaagtcttactacggtccaggtgacactgttgacacttccaagactttcactatcatcactcagttcaacactgacaacggttctccatccggtaacttggtttccatcactagaaagtaccagcagaacggtgttgatattccatctgctcaaccaggtggtgacactatttcctcttgtccatccgcttcagcttatggtggattggctactatgggaaaggctttgtcctctggaatggttttggttttctccatctggaacgacaactcccaatacatgaactggttggactctggtaatgctggtccatgttcttctactgagggtaacccatccaacatcttggctaacaacccaaacactcacgttgttttctccaacatcagatggggtgacattggttccactacaaactctactgctccaccaccaccacctgcttcctctactactttctccactactagaagatcctccactacttcttcctctccatcctgtactcaaactcactggggacaatgtggtggtattggttactccggttgtaagacttgtacttccggtactacttgtcagtactccaacgactactactcgcaatgccttgctctaga

Predict have with SEQ NO 1 be greater than 80%, preferably exceed 85%, more preferably exceed 90% and the nucleotide sequence that most preferably exceedes 95% identity modify.

According to another aspect of the present invention, provide nucleic acid construct, it comprises: the promotor of endo glucanase gene or expression; Multiple codons of Methionin and glycine residue; Nucleotide sequence with endo glucanase gene.Preferably, described endoglucanase promotor is the AOX1 promotor that derives from pPicz α A plasmid, and more preferably, described construct comprises pPicz α A plasmid.

Embodiment

In this detailed description, for illustration the present invention, provide the preferred embodiment about " thering is the biological activity protein of and immobilized focus crosslinked for orientation ", described embodiment is not intended to be limited in the scope defining in claims.

In this specific invention, the novel bioactive ring mutain compared with native protein with lasting stability has been described, its have be positioned at away from active centre place for the oriented immobilization of albumen and crosslinked focus.

Another aspect of this specific invention is to insert the method for genetically engineered these albumen, preferred enzyme by retaining ring, and described ring is for setting up for oriented immobilization and crosslinked focus.

The albumen of describing in this specific invention is selected from such albumen: it is any recombination mutation albumen, comprises enzyme, antibody, acceptor, antibody fragment, in conjunction with albumen and synthetic peptide.

" active centre " used herein can represent the region with catalytic group and substrate binding site.

" enzyme " used herein can represent, the polypeptide that catalytic molecular substrate transforms to the specificity of molecular product.

Bioactive mutain represents the mutain with interior ring of the function that retains native protein.The mutain that retains the general three structure of (except engineering ring) native protein is known as all essential tertiary structure element that comprises native protein.

Term used herein " mutant enzyme " is the enzyme that differs one or more features that caused by sudden change with natural enzyme.Term used herein " sudden change " is that the permanent heredity of gene or chromosomal nucleotide sequence changes.Term used herein " natural enzyme " can exchange and use with " wild-type enzyme ", and is the native state in it and by the enzyme of denaturing agent (such as the severe condition of heat, chemical substance, enzyme effect or extraction) change in cell.

" interior ring " used herein represents the structural element of the formation ring that comprises small peptide, and it is positioned at specifically the surface of folding mutain and is exposed to water.

Term " non-natural structural element " is non-existent structure in corresponding native protein as described herein.In view of described non-natural peptide ring does not exist in native protein, must use genetic modification technology by engineered its oligonucleotide sequence one-tenth target protein gene, thereby after protein expression, produce the mutain of expecting.In the mutain of expressing, interior ring structure has reflected that the peptide of specific formation ring adds to the interior type of the basic sequence of native protein.

" interior type interpolation " represents in this article, and mix described peptide in natural basic sequence at the appropriate point place between N and C terminal amino acid residue.Described appropriate point represent to be positioned at natural folding albumen lip-deep, along the arbitrary region of native protein basic sequence.The effect of mixing the engineered mutain basic sequence NX-PP-YC of expression of peptide, described basic sequence NX-PP-YC contains the described peptide PP that is positioned at native protein basic sequence NXYC.

N and C represent N and the C-terminal of basic sequence in this article, and X and Y have described the one or more amino-acid residues that are positioned at N and C end with regard to mixing site.Therefore, mutain can be regarded the peptide sequence that comprises whole wild-type sequence as, only extends the short block of the peptide of extra formation ring at certain non-distal point place.

" lasting stability " of albumen represents the lasting thermostability of folding albumen to antitypy and the folding albumen lasting stability to antidegenerative agent sex change as described herein.Term " sex change " represent the process of unfolding.Depend on condition, sex change can be reversible or irreversible.Proceed to comparatively high temps and can work the protein-denatured effect that makes, that is, and thermally denature.Denaturing agent can also be urea, Guanidinium hydrochloride, tensio-active agent, pH extreme condition and organic solvent.The folding functional protein of stablizing working as native protein under the same conditions also described in this term.By using term " condition ", should be appreciated that these conditions are that biology working conditions is such as pH and temperature.

" directed proteopexy " used herein can represent to change albumen from preselected site, can albumen being connected to surface by covalent force or noncovalent force.

Term used herein " focus " can represent to have in albumen the region of strong immobilization avidity.

According to another aspect of the present invention, provide the nucleotide sequence separating, it comprises the endo glucanase gene of modifying by adding multiple codons of Methionin and glycine residue by site-directed mutagenesis.Preferably, described gene is selected from the codon optimized form of Trichodermareesei (QM9414) egl1 gene.The nucleotide sequence of codon optimized Trichodermareesei (QM9414) egl1 gene provides as SEQ NO 1, and most preferably, described endo glucanase gene is by the nucleic acid sequence encoding of describing in SEQ NO 1.

According to another aspect of the present invention, provide nucleic acid construct, it comprises: the promotor of endo glucanase gene or expression; Multiple codons of Methionin and glycine residue; Nucleotide sequence with endo glucanase gene.Preferably, described endoglucanase promotor is the AOX1 promotor that derives from pPicz α A plasmid, and most preferably, described construct comprises pPicz α A plasmid.

In a preferred embodiment of the invention, oligonucleotide sequence described herein is characterised in that, the insertion point that it comprises the interior ring sequence for insert surface exposure via site-directed mutagenesis, and preferably, described insertion point be in folding albumen coding away from amino acid whose any site in active centre.

In another preferred embodiment of the present invention, oligonucleotide sequence, wherein insertion point is positioned on the almost offside of 180 ° in active centre of existing ring, and more preferably, insertion point is positioned at the come-at-able and flexible region of height solvent in existing ring region.Most preferably, insertion point is between the 345th and the 346th base.

Oligonucleotide sequence is characterised in that, it comprises the interior ring sequence that uses surface that overlapping PCR extension method is introduced by site-directed mutagenesis to expose.By the method, preliminary PCR can overlap constant gene segment C, then used as the template DNA of another PCR to set up full length product.

Therefore, oligonucleotide sequence is characterised in that, it comprises the interior ring sequence that uses surface that overlapping PCR extension method is introduced by site-directed mutagenesis to expose.Conventionally 4 kinds of primers of application and preferred 3 PCR reaction cycle.

In a preferred embodiment of the invention, oligonucleotide sequence is characterised in that, it comprises the interior ring sequence that uses the overlapping PCR extension method surface of introducing by site-directed mutagenesis to expose, described overlapping PCR extension method uses 4 kinds of primers and preferred 3 PCR reaction cycle, and most preferably, primer 1 (5 ' CCG cCAGCAACCGGGTACCAGCACC3 ') complementary and contain EcoRI Restriction Enzyme cutting site (be shown as bold-type letter and indicate underscore) with 5 ' end of egl1 gene; Primer 2 (5 ' gTCcAAGTACAATCTTG3 ') and the interior ring insertion sequence (5 ' ACCTTTCTTACCCTTCTTCTTACCCTTCTT3 ') (be shown as bold-type letter and indicate underscore) that exposes of the surface complementary and that contain encoded K KGGKKKGGK of 17 Nucleotide between the 329th and the 345th base of egl1 gene; Primer 3 (5 ' tCCGACGGTGAATACGGT3 ') and the interior ring insertion sequence (5 ' AAGAAGGGTAAGAAGAAGGGTAAGAAAGGT3 ') (be shown as bold-type letter and indicate underscore) that exposes of the surface complementary and that contain encoded K KGGKKKGGK of 18 Nucleotide between the 346th and the 364th base of egl1 gene; Primer 4 (5 ' CCG gCAAGGCATTGCGAGTAGTAG3 ') complementary and contain XbaI Restriction Enzyme cutting site (be shown as bold-type letter and indicate underscore) with 3 ' end of egl1 gene.

At gene level, the oligonucleotide that forms the ring of surface exposure represents such oligonucleotide sequence: it is designed to, and after expressing, forms the peptide ring structure of the come-at-able surface of water that is positioned at mutain.Described oligonucleotide must be genetically engineered in suitable site process along wild-type exon.The site of selecting for genetic manipulation must be such site: it produces natural sample protein structure after mutator gene is expressed.Natural sample protein structure represents three grades of such (totally) structures: its must structural element with native protein all and one (or may be multiple) extra be positioned at lip-deep ring structure.In brief, must not can disturb and transcribe normally rear and translation post-treatment event for the site of genetic manipulation selection, and it must allow protein folding to follow natural approach, thereby produce the mutain with natural spline structure and (or may be a multiple) extra loop.The concept in " insertion " and " site " is consistent with the work of initial use Restriction Enzyme and ligase enzyme, is cut off completely thus along the site of DNA, inserts extra DNA fragmentation, and connects described fragment.With regard to adopting the invention of overlapping PCR elongation technology, oligonucleotide seems to insert in natural exon at specific site again, therefore causes the application of such term.But the mechanism that realizes such variation is diverse, and depend on archaeal dna polymerase and the suitably application of the DNA primer of selection.Therefore, term is limited to scope and the background of overlapping PCR elongation technology such as " site " and " insertion " application in the present invention.

Term used herein " overlapping PCR extends " can represent such method: it allows use polymerase chain reaction and do not use restriction endonuclease or T4 DNA ligase, and oligonucleotide sequence is inserted in the oligonucleotide sequence of selecting.Initial PCR can overlap constant gene segment C, and then described constant gene segment C is used as the template DNA of another PCR to set up full length product.Inner primer 3 ' end that overlap on centre portion, complementary, and introduce nucleotide subsitution, insertion or disappearance (for site-directed mutagenesis or for gene splicing), be coded in the Nucleotide of finding in abutting connection with the connection section place of constant gene segment C.The overlapping chain of these intermediate products in PCR subsequently in this 3 ' location hybridization, and being extended to produce the full length product by side joint primer amplification, described full length product can comprise that Restriction Enzyme site is for inserting described product for cloning the expression vector of object.

" exon " used herein can represent, after removing the intron of precursor RNA by montage, and the nucleotide sequence (DNA or RNA) being represented by the mature form of RNA molecule.Ripe RNA molecule can be the functional form of messenger RNA(mRNA) or non-coding RNA (such as rRNA or tRNA).

Term used herein " genetic expression " can represent, the process by the information from gene for the synthesis of functioning gene product.These products are often albumen.

Term used herein " hybridization " can represent that the complementary base pairing of a nucleic acid and another nucleic acid interacts, and it causes the formation of duplex, triplex or other high stage structure, and uses interchangeably with " annealing " in this article.

" overlapping end " used herein can represent such oligonucleotide sequence: it is complimentary to one another, and is positioned at 5 ' or 3 ' end of oligonucleotide sequence.These oligonucleotide sequences 5 ' or 3 ' end comprise and insert insertion sequence corresponding to ring section.

Therefore,, by using term " at least 60% identity ", should be appreciated that the present invention not only comprises the application of concrete Exemplary core nucleotide sequence.Also predict the modification to described sequence, such as the disappearance in sequence, insertion or displacement, its:

A) produce " silence " and change, it can not affect in fact the functional performance of protein molecular.For example, predict the degeneracy of reflection genetic code or cause producing the equivalent amino acid whose nucleotide sequence change of chemistry in given site, or

B) promote active improvement or the modification of substrate specificity.

Term used herein " codon " is the Nucleotide of one group of 3 vicinity, and also referred to as triplet, it is given for the synthetic amino acid whose type of albumen and order.Term " promotor " is the DNA region of transcribing that promotes specific gene as described herein.

The endoglucanase i gene of Trichodermareesei is the template for site-directed mutagenesis.To encircle and insert in this gene by site-directed mutagenesis.The ring inserting in endoglucanase i gene is for generation of the mutator gene that inserts the endoglucanase i albumen encircling.In pichia pastoris phaff (Pichia pastoris), this gene is expressed on restructuring ground.The endoglucanase i albumen that inserts ring is by the product for oriented immobilization or crosslinked research.

In a preferred embodiment of this concrete invention, 4 kinds of different 10 amino acid whose long rings (being made up of Methionin and glycine residue) are introduced in endoglucanase i (EGI) to the i.e. main endoglucanase of Trichodermareesei.In the present invention, find that EGI_L5 is the most stable ring mutant enzyme.Successfully in pichia pastoris phaff, express and purified mutant body endoglucanase i (EGI_L5) and natural endoglucanase i (EGI).The stability of intentional sudden change on enzyme and active impact are analyzed.

Materials and methods

microorganism, enzyme and chemical substance:

Use intestinal bacteria (Escherichia coli) Top10F as the host for amplification vector and subclone.By pichia pastoris phaff KM71H (aox1:ARG4, arg4) (Invitrogen, SanDiego, USA) for expression of recombinant proteins.By pPICZ α A carrier (Invitrogen, San Diego, USA) for clone and protein expression.PPICZ α A plasmid is containing the alpha factor secretion signal, alcohol oxidase (AOX) promotor of expressing for the Zeocin resistant gene selected intestinal bacteria, for the methanol induction of the gene of cloning of cell exocrine of gene product that is useful on clone.By Pfu polysaccharase and Rapid Ligation Kit (Fermentas) for cloning object.By Taq polysaccharase (Qiagen) for bacterium colony PCR.All chemical substances that comprise are all AGs.

protein determination:use BCA protein determination reagent (Pierce) to determine protein concentration.Bovine serum albumin is used as to protein standard substance.

enzymatic determination:carry out in triplicate all CMC determinations of activity, there is the standard deviation lower than 10%.Carry out in duplicate or in triplicate all 4-MUC determinations of activity, there is the standard deviation lower than 10%.

Provide following embodiment to carry out illustration the present invention, and be not intended to limit the scope of the invention.

embodiment 1:

The site-directed mutagenesis of endoglucanase:

Apply overlapping PCR extension method and introduce ring sudden change.According to document people such as (, 1994) Vallejo, design overlapping PCR and extend primer (forward egl1:5 ' CCG gAATTCcAGCAACCGGGTACCAGCACC3 ', oppositely egl1:5 ' CCG tCTAGAgCAAGGCATTGCGAGTAGTAG3 ', the overlapping extension primer of forward: 5 ' aAGAAGGGTAAGAAGAAGGGTAAGA aAGGtTCCGACGGTGAATACGGT3 ', reverse overlapping extension primer: 5 ' aCCTTTCTTACCCTTCTTCTTACCCTTCTTgTCCAAGTACAATCTTG3 ').Ring sudden change is introduced between the 112nd and the 113rd amino acid.Confirm the fidelity of reproduction of construct by order-checking.

embodiment 2:

The clone of egl1 and modified egl1 (egl1_L5) gene

Obtain carrying at each arm codon optimized (for pichia pastoris phaff) endoglucanase 1 synthetic gene (egl1) of EcoRI and XbaI restriction site from GeneART.The protein sequence of synthetic gene is identical with trichoderma reesei endoglucanase 1 (GenBank registration number M15665).EcoRI and XbaI site are added into gene side joint region.By EcoRI and XbaI digestion for synthetic gene, and use Rapid Ligation Kit (Fermentas) to reconnect in the EcoRI-XbaI site of pPicz α A carrier.Egl1 subclone is entered in intestinal bacteria Top10F cell.Have 25 μ g/ml zeocin exist under on less salt LB flat board, cultivate Bacillus coli cells.Select the bacterium colony of the Zeocin positive, put into PCR test tube, and carry out bacterium colony PCR and react as normal PCR with 5 ' AOX (5 '-GACTGGTTCCAATTGACAAGC-3 ') and 3 ' AOX (5 '-GCAAATGGCATTCTGACATCC-3 ') primer, but carry out main denaturing step 10 minutes instead of 5 minutes at 95 DEG C.Select the positive bacterium colony of bacterium colony PCR, and use MiniPrep Kit (Qiagen) to separate recombinant plasmid.

embodiment 3:

The conversion of mutant enzyme and screening:

Before transforming, by recombinant plasmid pPICz α A-egl1 SacI linearizing.This process causes one or more copies of linearizing carrier to enter in 5 ' the AOX1 chromogene seat of pichia pastoris phaff KM71H by homologous recombination stable integration.All transformant that obtain are mutant (slowly utilizing the phenotype of methyl alcohol).According to the method (Wu and Letchworth, 2004) of combinatorial chemistry conversion and electroporation, preparation competence pichia pastoris phaff KM71H cell.By the linearizing recombinant plasmid of approximately 1 μ g and competence KM71H cytomixis.Immediately mixture be transferred in the 0.2cm electroporation cuvette of precooling and hatch on ice 5 minutes.After electroporation, immediately 1M sorbyl alcohol ice-cold about 1ml is added in cuvette.Charging voltage, electric capacity and resistance are respectively 1.5kV, 25F and 200.Transformation mixture is spread on the YPD flat board that contains 100 μ g/ml zeocin.Flat board is hatched at 30 DEG C, until bacterium colony occurs and growth (approximately 3 days).Select the bacterium colony of the Zeocin positive, put into PCR test tube, described test tube microwave treatment is also placed on ice-water bath immediately, and adds the cold ddH of 10 μ l to each test tube ₂o, the template by 1 this cell mixture of μ l as bacterium colony PCR.Use 5 ' AOX and 3 ' AOX primer to carry out bacterium colony PCR.Select the bacterium colony of the bacterium colony PCR positive.

Containing blue azo-group Cellulose,ether with glycolic acid (Azo-CMC) (0.25%w/v) as minimum methyl alcohol agar (the Buffered Minimal Methanol Agar of the buffering of substrate, BMM-agar) dull and stereotyped (100mM potassiumphosphate, pH6.0,1.34%YNB, 4 × 10-5% vitamin H, 0.5% methyl alcohol) the upper multiple copied transformant of selecting.According to the relative radius in the transparent area in periphery of bacterial colonies, select the transformant of more activated expression enzyme.In the time that described enzyme has activity, its Azo-CMC that can degrade, and as the result of enzymically hydrolyse and produce transparent area in periphery of bacterial colonies.

embodiment 4:

The small-scale of Recombinant Pichia pastoris bacterial strain is expressed:

The pichia pastoris phaff recombined bacterium of producing endoglucanase i and its ring mutant is fallen to inoculating into 50mL BMG substratum (100mM potassiumphosphate, pH6.0,1.34%YNB, 4 × 10-5% vitamin H and 1% glycerine) in, and spend the night in 30 DEG C of cultivations (250rpm) in the baffled shaking flask of 250ml.In the time that OD600 reaches 10 units/ml, carry out collecting cell by centrifugal (3000 × g, 5min).With the initial OD600 of 30 units/ml, cell precipitation thing is suspended in BMM substratum (containing 0.5% (w/v) methyl alcohol to substitute the BMG of 1% glycerine) again.Culture is cultivated approximately 120 hours at 30 DEG C.Within every 24 hours, add the final concentration of methyl alcohol to 10g/L.Every 24 hours collecting cell culture supernatants.

embodiment 5

General fermentation process:

EGI clone E12 and EGI_L5 clone D5 are carried out to fed-batch fermentation.In the second section of fermentation, add glycerine to promote the growth of Recombinant Pichia pastoris culture.Different time points is during the fermentation collected tunning, and carries out SDS-PAGE and activation analysis for the tunning of collecting in different time points.After clone's growth velocity, measure and calculate the cell dry measure (CDW) of each sample of collecting in different time points.For each sample, form speed (mM) according to the 4-MU of per minute, evaluate the activity for 4-MUC.Use the specificity methyl alcohol probe in fermentor tank, monitoring is at the methanol concentration of each fermentation time point.Produce the clone's of EGI and EGI_L5 fermentation data analysis instruction, find that EGI and EGI_L5 growth velocity and express spectra in fed-batch fermentation process are very similar.Find that enzymic activity and production of enzyme are along with methanol concentration increases and increases.

As expected, do not produce enzyme at the glycerine of two kinds of fermentations in batches with in the glycerine fed-batch stage.Within 24 hours, start to produce EGI by methanol induction AOX promotor later, started to produce EGI_L5 at 20 hours later.In methanol feeding batch phase, Growth of Cells is minimum.The activity of the recombinase of every kind of production is maximum in the time of fermentation ends.

embodiment 6:

The bioreactor culture of Recombinant Pichia pastoris bacterial strain:

With the initial volume of 2L, with the feed rate of 18ml/h/L glycerine and 1-12ml/h/L methyl alcohol, in 7.5L fermentor tank (BioFlo110, New Brunswick Scientific), cultivate with pH5 the pichia pastoris phaff clone who carries endoglucanase 1 and its ring mutant at 28 DEG C.Use the fermention medium prescription of Invitrogen Corp. to carry out fed-batch fermentation together with PTM1 trace-metal solution.Fermentation glycerine in batches with the glycerine fed-batch stage in, use glycerine as sole carbon source.In the methanol feeding batch phase of fermentation, use the inductor of methyl alcohol as AOX promotor.Use specific methyl alcohol probe (Raven Biotech), monitoring methyl alcohol level.Tunning is filtered, buffer exchange, and use Sartocon Micro and Ultrafiltration System (Sartorius-Stedim) to concentrate.Use has the Sartocon Slice200HydroSart film of 0.45 micron of cutoff and has 100kDa and the polyethersulfone of 10kDa cutoff (PES) film.

embodiment 7:

The tryptic limit protein enzymolysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme spectrum analysis and use:

Use zymogram gels is analyzed, the activity of the recombinase that monitoring is produced qualitatively.By the substrate performing an analysis for 4-MUC.The EGI that produces of finding to recombinate in pichia pastoris phaff has and exceedes one to the activated protein band of 4-MUC tool (50,70 and 100kDa).Think that these are different glycosylation (70kDa) and/or dimerization products (100kDa).Trypsinase is indicated the limit protein enzymolysis of EGI albumen, and 50kDa product exists as the result of proteolysis degraded.EGI_L5 shows the more low activity to 4-MUC in enzyme spectrum analysis.Also finding that it has exceedes a kind of enzymatic activity composition (wherein about 100kDa product is advantage product) and a kind of possible glycation product (about 70kDa).Find that two kinds of forms all have activity to 4-MUC.

With 5% (w/v) stacking gel and 12% (w/v) separating gel, by cell culture supernatant swimming on SDS-PAGE gel of collecting.About 15-20 μ l supernatant liquor is loaded in each hole of gel.After electrophoresis, gel is dyeed with coomassie brilliant blue R_250.For enzyme spectrum analysis, carry out natural SDS-PAGE.Gel is each 10 minutes with 2.5% (v/v) TritonX-100-50mM sodium acetate soln (pH4.8) washing 3 times.Then, by gel with 50mM sodium-acetate buffer (pH4.8) washing 3 times each 10 minutes.Use 4-MUC (4-methyl umbrella shape base β-D-cellobioside) to carry out enzyme spectrum analysis as substrate.The solution of the 4-MUC that uses 1ml0.5mg/ml in enzyme spectrum analysis in 50mM sodium-acetate buffer (pH4.8).Under ultraviolet ray, develop and record enzymic activity.After enzyme spectrum analysis, gel is dyeed with coomassie brilliant blue R_250.Use Gel-Doc (BioRad) to record all gel photograph.Use protein groups classes and grades in school trypsin Sigma), on the EGI of purifying albumen, carry out limit protein enzymolysis.Trypsinase is dissolved in to the final concentration to 0.02 μ g/ μ l in 50mM acetic acid.0.02 μ g/ μ l trypsinase is added in the EGI enzyme in 50mM ammonium bicarbonate buffers (pH7.8), makes the trypsinase in sample: the ratio of albumen is 1:50 (w/w).Digestion reaction thing is hatched 75 minutes at 37 DEG C, and collect sample in different time points (0-5-10-15-30-45-60-75 minute).By taking out the aliquots containig of sample and making sample swimming on SDS-PAGE gel, monitor proteopeptic degree.

embodiment 8:

The purifying of recombinant protein:

With RAC in batches after affinity purification, by two kinds of enzyme purifications to homogeneity.The protein band of 2 approximately 70 of purifying and 100kDa from the rough protein solution of EGI, and purify the only glycosylation form of a kind of about 70kDa from the rough protein solution of EGI_L5 with RAC.In other glycation product of EGI and EGI_L5, the component of purifying is the most activated product.

Use the amorphous cellulose (RAC) of regeneration as affinity chromatography matrix, purifying EGI and EGI_L5.Obtain the affinity purification in batches of two kinds of enzymes.Amorphous cellulose by Avicel Ph105 for the synthesis of regeneration.Carry out purifying in room temperature.Spent glycol wash-out recombinant fiber element enzyme, and with 50mM sodium-acetate buffer (pH5) by further remove and exchange ethylene glycol (due to its interference to BCA protein determination) through the ultrafiltration of 10kDa cutoff Vivaspin500 film revolving filter (Sartorius-Stedim).

embodiment 9

Confirm the experiment of the impact of temperature on enzymic activity

By 3,5-dinitrosalicylic acid (DNS) method, in 50mM sodium-acetate buffer (pH4.8), determine every kind of enzyme in differing temps (15 DEG C-95 DEG C) activity (Ghose, 1987) to 0.5%CMC (w/v).Every kind of enzyme and substrate peak are measured to temperature preincubate 5 minutes at every kind.Enzyme and substrate are hatched 10 minutes in mensuration temperature.Measure the reducing sugar producing at 550nm.Glucose is used as to standard substance, and according to IUPAC method, enzymic activity is calculated as to CMC unit/ml, for measuring cellulase activity (Ghose, 1987).By using enzyme at the maximum activity of definite temperature as 100%, calculate residual enzyme activity.

Use DNS method and the 0.5%CMC (w/v) as substrate, definite EGI producing by fermentation and the temperature activity profile of EGI_L5.Two kinds of recombinases show similar spectrum in the activity of differing temps.EGI shows maximum activity at 45 DEG C and 55 DEG C, and finds that EGI_L5 has maximum activity at 35 DEG C.Two kinds of enzymes show activity in wide temperature range (15 DEG C to 65 DEG C).Although EGI_L5 is 55 DEG C of activity with reduction compared with EGI, it shows higher a little relative activity at 65 DEG C, 75 DEG C and 85 DEG C.Two kinds of enzymes keep the active approximately 40% of them at 75 DEG C, and keep their active 30% at 85 DEG C.

embodiment 10

Confirm the experiment of the impact of pH on enzymic activity

Use 3,5-dinitrosalicylic acid (DNS) method, determine in different pH damping fluids at 55 DEG C every kind of enzyme at different pH (pH3-6) to the activity of 0.5%CMC (w/v) 10 minutes.By Citrate trianion-phosphate buffered saline buffer of pH3 (100mM citric acid, 200mM Na ₂hPO ₄), the 50mM sodium phosphate buffer of the 50mM sodium-acetate buffer of pH4 and pH5, pH6 is for determining the impact of pH on enzymic activity.Measure the reducing sugar producing at 550nm.Glucose is used as to standard substance.By using enzyme at the maximum activity of definite pH as 100%, calculate residual enzyme activity.

Two kinds of enzymes all show maximum activity at pH5.Observe the pH activity profile of two kinds of enzymes and follow very similarly spectrum.Find that enzyme is non-activity almost at pH3.Find that EGI and EGI_L5 enzyme show the kinetics of Michaelis-Menten type to the substrate 4-MUC of solubility.Determine the kinetic constant (Km and kcat) at 45 DEG C.The Km value of finding EGI and EGI_L5 is respectively 0.47mM and 0.54mM.Although Km value is that similarly EGI and EGI_L5kcat value exist significant difference.Find that EGI has 0.013 second ^-1kcat value, and EGI_L5 has shown 0.004 second ^-1kcat value.

embodiment 11

4-MUC measures

According to people such as (, 1989) Chernoglazov, the 4-MUC that carries out tunning measures.Fermented sample is hatched in 50mM sodium-acetate buffer (pH4.8) together with 0.5mg/ml4-MUC.Carry out dynamic analysis 30 minutes at 45 DEG C.4-methyl umbelliferone (4-MU) is used as to standard substance.The 4-MU discharging with fluorescence spectrophotometer measurement, excites and launches at 435nm at 363nm.Enzymic activity is calculated as to the RFU of per minute release or the 4-MU (mM) that per minute discharges.In 50mM sodium-acetate buffer (pH4.8), determine EGI and the EGI_L5 albumen kinetic constant (kcat and Km) for 4-MUC at 45 DEG C.By 6 kinds of different concentration of substrate (50-2000 μ M) for kinetic determination.In mensuration, enzyme concn is remained on to 0.3 μ M.Carry out in duplicate all measurements.By by the program of OriginPro by initial speed data matching to Michaelis-Menten equation, computational dynamics constant K m and kcat.

embodiment 12

Stability Determination

First hatch 10 minutes at 100 DEG C with every kind of enzyme of about 0.4mg/ml, determine enzyme stability.Then by enzyme cooled on ice 10 minutes.100 DEG C of that hatch and enzyme samples that do not hatch (at 55 DEG C and pH4.8) are carried out to standard C MC and measure 10 minutes.Calculate the stability of every kind of enzyme with respect to its normal activity with the form of retentive activity.

Hatch 0h to 72h by two kinds of enzymes with about 0.4mg/ml at 50 DEG C, and be determined at 50 DEG C and pH4.8 to the enzymic activity of 1%CMC (w/v) 10 minutes, determine the residual enzyme activity at 50 DEG C.Using the enzymic activity of not hatching at 50 DEG C (time 0) as 100%.Hatch for every kind, carry out at least 2 independent activity tests.At each incubation time, measure in triplicate recombinase, there is the standard deviation lower than 10%.

Also hatch 0h to 2h by two kinds of enzymes with about 0.4mg/ml at 70 DEG C, and be determined at 50 DEG C and pH4.8 to the enzymic activity of 1%CMC (w/v) 10 minutes, determine the residual enzyme activity at 70 DEG C.Using the enzymic activity of not hatching 50 DEG C of mensuration (time 0) as 100%.Determine in triplicate enzymic activity, there is the standard deviation lower than 10%.

100 DEG C hatch 10 minutes after, find that EGI and EGI_L5 retain respectively their active 94.76% and 95.41%.The EGI calculating with ExPASy people such as (, 1993) Bjellqvist and the pI of EGI_L5 are respectively 4.66 and 5.35.Although introducing 10 amino acid whose rings in the situation that, the pI of the EGI of calculating almost 1 the pH unit of having drifted about, its pH spectrum does not change.50 DEG C hatch 72 hours after, show that EGI and EGI_L5 keep their active 65% and 57% at 50 DEG C respectively.In addition, confirm EGI 50 DEG C hatch for a long time after (after 24h to 72h) lose more quickly its activity.In addition, two kinds of enzymes hatch at 70 DEG C the similar spectrum that has shown their residual enzyme activity for 2 hours later.EGI has shown 5.3% of residual enzyme activity and has declined, and EGI_L5 is hatched and within 2 hours, lost its active 18% later at 70 DEG C.

Claims (22)

1. bioactive mutain, the non-natural structural element that all essential tertiary structure element that it comprises corresponding native protein and at least one surface expose, it is characterized in that, described non-natural structural element is positioned at albumen primary structure and forms the peptide ring that surface exposes, and described peptide ring is for setting up focus to allow the enhanced stability of directed proteopexy and folding albumen tertiary structure.

2. bioactive mutain according to claim 1, wherein said mutain is selected from and comprises enzyme, antibody, acceptor, antibody fragment, albumen in conjunction with albumen and synthetic peptide.

3. bioactive mutain according to claim 2, preferably enzyme of wherein said mutain, more preferably cellulase.

4. bioactive mutain according to claim 3, wherein said mutain is endoglucanase.

5. bioactive mutain according to claim 4, wherein endoglucanase enzyme is the extract of Trichodermareesei (trichoderma reesei).

6. according to bioactive mutain in any one of the preceding claims wherein, the peptide ring that the surface of wherein said insertion exposes comprises at least 5 and 15 amino acid at the most.

7. according to bioactive mutain in any one of the preceding claims wherein, the peptide ring that the surface of wherein said insertion exposes is the translation after product of the oligonucleotide sequence that comprises modified endonuclease gene, and described endonuclease gene has multiple codons of the aminoacid sequence that is selected from Methionin and glycine.

8. bioactive mutain according to claim 7, the peptide ring that the surface of wherein said insertion exposes comprises Methionin and glycine aminoacid sequence alternately.

9. bioactive mutain according to claim 7, wherein said endonuclease gene comprises the nucleotide sequence with SEQ NO 1 with at least 60% sequence identity.

10. bioactive mutain according to claim 7, the peptide ring that the surface of wherein said insertion exposes is defined as KKGGKKKGGK.

11. according to bioactive mutain in any one of the preceding claims wherein, and the peptide ring that the surface of wherein said insertion exposes is positioned on the offside of 180 ° substantially in active centre of described albumen, in the come-at-able and flexible existing ring of solvent.

12. bioactive mutains according to claim 11, the peptide ring that the surface of wherein said insertion exposes is positioned at apart from the C α of active centre residue approximately the 112nd and the 113rd residue between.

The method of the bioactive mutain of describing in 13. genetically engineered claims 1, described method comprises the steps:

(1) limit site along the exon region of target protein gene, described site is used for inserting the oligonucleotide of the ring that forms surface exposure,

(2) sequence of restriction DNA primer, described sequence comprises the overlapped ends that forms the oligonucleotide bridging encircling by described, and described primer is designed to the area hybridization along the insertion point of described selection via described overlapped ends,

(3) chemical synthesising DNA primer, described DNA primer comprises the described overlapped ends that forms the oligonucleotide sequence bridging encircling by described,

(4) apply overlapping PCR and extend, to prepare the mutator gene of the oligonucleotide sequence that contains described formation ring, described oligonucleotide sequence is positioned at the designated area of the exon of target protein gene,

(5) application DNA separates with mutator gene described in purifying,

(6) express described mutator gene, and

(7) mutant of the described target protein of the peptide ring that separation contains the exposure of described surface.

14. methods according to claim 14, wherein said oligonucleotide sequence comprises the nucleotide sequence with SEQ NO 1 SEQ NO 1 with at least 60% sequence identity.

15. oligonucleotide sequences, the nucleotide sequence that it comprises modified endo glucanase gene, described modified endo glucanase gene has multiple codons of the aminoacid sequence that is selected from Methionin and glycine.

16. oligonucleotide according to claim 15, wherein endonuclease gene comprises Methionin and glycine aminoacid sequence alternately.

17. oligonucleotide according to claim 15, wherein said endonuclease gene is selected from (QM9414) egl1 gene of Trichodermareesei (Trichoderma reesei).

18. oligonucleotide according to claim 15, wherein said endo glucanase gene is by following sequence encoding: the nucleotide sequence of describing in SEQ NO 1, the reverse complement of described sequence, the complement of described sequence, the reversion of described sequence, or with aforementioned sequence in any nucleotide sequence there is the sequence of at least 60% sequence identity.

19. nucleic acid constructs, it comprises: the promotor of endo glucanase gene or expression, described endo glucanase gene comprises the nucleotide sequence with SEQ NO 1 with at least 60% sequence identity; Multiple codons of Methionin and glycine residue; Nucleotide sequence with described endo glucanase gene.

20. nucleic acid constructs according to claim 19, wherein said promotor comprises pPicz α A plasmid.

21. nucleic acid constructs according to claim 19, wherein in pichia pastoris phaff (Pichia pastoris) host, and most preferably in pichia pastoris phaff KM71 H bacterial strain, express pPicz α A plasmid.

22. nucleotide sequences, itself and SEQ NO 1 have at least 60% sequence identity.