CN102206625A

CN102206625A - Orthogenesis of protein introns through error-prone PCR/kanamycin screening system

Info

Publication number: CN102206625A
Application number: CN2011100532438A
Authority: CN
Inventors: 李曼; 孟清
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2011-03-04
Filing date: 2011-03-04
Publication date: 2011-10-05

Abstract

The invention relates to a method for orthogenesis of protein introns through an error-prone PCR (polymerase chain reaction)/kanamycin screening system, comprising the steps of: 1) preparing lots of carriers used for orthogenesis; 2) conducting an error-prone PCR to the protein introns which are then connected to the carriers used for orthogenesis and inactivated, transforming competent cells through condensation, and coating with a bi-anti-plate containing ampicillin and kanamycin; 3) performing a bacteria liquid PCR verification to bacterial colonies growing on the bi-anti-plate, obtaining mixed plasmids of preliminarily identified positive clones as the template of next error-prone PCR, carrying out multiple rounds of error-prone PCR, thus obtaining evolved protein introns. The method of the invention can agilely screen protein introns with improved splicing activity. By adjusting the proportion of each component in a PCR system, lots of DNA fragments are amplified. Protein introns obtained through the method of the invention are high in splicing activity.

Description

Fallibility PCR/ kantlex screening system directed evolution of proteins intron

Technical field

The invention belongs to directed evolution of proteins intron field, particularly a kind of method of fallibility PCR/ kantlex screening system directed evolution of proteins intron.

Background technology

Protein intron is distributed in archeobacteria, bacterium and the eubacterium.It is the one section polypeptide chain that is present in the precursor protein, cut and to be the albumen exon with its both sides sequence be connected to form the mature protein of function with ormal peptide link in protein translation after ripening process, this process is called the protein splicing of protein intron mediation.The encoding sequence of albumen intron is positioned in the middle of the albumen exons coding sequence of both sides, successive open reading frame of they common formations, the translation back forms a precursor protein, and protein intron makes natural peptide bond (see figure 1) of its two exon intermediate formation in outside by the protein splice process.

Protein splicing is proved to be the important pivot of numerous protein engineerings.At present, protein intron has been successfully applied to proteinic modification and mark, and cyclisation is proteinic synthetic, the production of toxin protein, and proteinic fragment label is used for the protein synthesis of NMR research and viable cell etc.But when natural protein intron was inserted in the nonhost protein, most protein introns partially or completely lost the montage activity, had had a strong impact on protein intron as the instrument of protein splicing and be widely used.

Based on the limited understanding to the protein intron structural information, the montage activity that the method by orthogenesis improves protein intron is an approach preferably.The advantage of this method be do not need to understand protein intron three-dimensional structure information and with the proteinic mechanism of action of montage, in-vitro simulated process of evolving naturally makes the protein intron gene that a large amount of variations take place, and orthoselection goes out the protein intron of required character or function.The strategy of orthogenesis has fallibility PCR (polymerase chain reaction), DNA shuffling, and staggered extension, gradual cutting produces methods such as heterozyme.The key of orthogenesis technology is to set up a screening system efficiently.At present, beta-galactosidase enzymes, GFP, APH often are utilized the reporter protein as protein intron montage screening system.But the present domestic protein intron that does not also have the orthogenesis success.

Summary of the invention

Technical problem to be solved by this invention provides a kind of method of fallibility PCR/ kantlex screening system directed evolution of proteins intron, this method can be screened the active protein intron that improves of montage more delicately, by adjusting the proportioning of each composition of PCR system, amplification a large amount of dna fragmentation that comes out, the protein intron montage that the present invention obtains is active high.

The method of a kind of fallibility PCR/ kantlex screening system directed evolution of proteins intron of the present invention comprises:

1) utilize a pair of primer (5 ' end adds II type restriction enzyme Bsa I) that carrier pKH is carried out inverse PCR (polymerase chain reaction), then with inverse PCR product phosphorylation, from connecting transformed competence colibacillus, obtain plasmid pKH ', the BsaI enzyme is cut the fragment that 3516bp is reclaimed in the back, promptly obtains to be used in a large number the carrier of orthogenesis;

2) utilize a pair of primer (5 ' end adds II type restriction enzyme Bsa I) that protein intron is carried out fallibility PCR, be connected with the above-mentioned carrier that is used for orthogenesis then and deactivation, again through concentrating the transformed competence colibacillus cell, be coated with the two anti-flat board of the kantlex that contains ammonia benzyl mycin and lower concentration; Wherein fallibility PCR reaction conditions is as shown in table 1, and fallibility PCR comprises the Mg of basic, normal, high concentration ²⁺, Mn ²⁺And dXTP, totally 12 reactions, the amount of template is more than the amount of normal pcr template among the fallibility PCR, is 50-100ng;

Table 1 fallibility PCR reaction conditions

3) bacterium colony of growing is carried out bacterium liquid PCR checking on above-mentioned two anti-flat boards, and obtain it with the positive clone of preliminary evaluation and mix the template of plasmid as next round fallibility PCR, carry out 4-6 wheel fallibility PCR, a highest protein intron of montage activity of acquisition is the protein intron after the evolution that obtains by orthogenesis.

Above-mentioned steps 1) a pair of primer described in is primer PKHA and PKHB; it comprises protection base (ATC), and the BsaI enzyme is cut sequence (dash area), inserts four bases (small letter part) of fragment CP end; and with carrier complementary sequence (line part), as shown in Figure 3.

Above-mentioned steps 2) a pair of primer described in is primer CPb1 and CPb2, and it comprises protection base (ATC), and the BsaI enzyme is cut sequence (dash area), inserts fragment complementation sequence (line part), as shown in Figure 3.

Above-mentioned steps 2) protein intron described in is a Cne PRP8 protein intron.

The mol ratio of inserting fragment and carrier when connecting above-mentioned steps 2) is 3: 1.

Above-mentioned steps 3) in every take turns fallibility PCR after, improve the concentration of the kantlex in two anti-flat boards, till the colony number on two anti-flat boards no longer increases, obtain the higher protein intron of montage efficient, and verify with the Western trace whether its montage efficient improves.

The present invention is by carrying out many wheel fallibility PCR to the protein intron that inserts among the pKH, and the kalamycin resistance screening constantly accumulates useful sudden change, thereby obtains the active high protein intron of montage.

The kantlex screening system is realized by carrier pKH.The gene that contains the kanR that encodes among the pKH, the albumen of the anti-kantlex of can encoding.Protein intron is inserted in the gene of kanR,, thereby can contain on the flat board of kantlex and grow if the protein intron generation montage of orthogenesis then forms corresponding proteins matter intron and the albumen of the active anti-kantlex of function is arranged.In the orthogenesis process, adopted many wheels fallibility PCR, each template of taking turns fallibility PCR is the positive colony of last round of orthogenesis, and improving constantly the screening concentration of kantlex, the so constantly positive sudden change of accumulation makes the protein intron that is obtained have higher montage efficient.The height of protein intron montage efficient is by the checking of Western trace.

The key of orthogenesis success mainly contains three: (1) screening system, the present invention has used the kantlex screening system, screens the active protein intron that improves of montage more delicately.(2) needs obtain the sudden change storehouse of amounts of protein intron, have comprised the fallibility PCR environment of low middle and high concentration in the error-prone PCR systems among the present invention, adjust the proportioning of each composition of PCR system in addition, and the dna fragmentation amount that amplification is come out is many.Mol ratio the best when (3) the insertion fragment is connected with carrier, the ratio among the present invention is 3: 1.

Beneficial effect

1, the present invention has used the kantlex screening system, can screen the active protein intron that improves of montage more delicately, and optimize linked system, makes that the mol ratio of inserting fragment and carrier is 3: 1, thereby reaches best joint efficiency.

2, of the present invention simple to operate, lower to equipment requirements, and the protein intron montage that this method obtains is active high.

Description of drawings

Fig. 1 is a protein intron montage mechanism.

Fig. 2 is orthogenesis scheme synoptic diagram (is example with Cne PRP8).

Fig. 3 is primer sequence figure among the present invention.

Fig. 4 is a fallibility PCR picture.

Fig. 5 for Cne PRP8 protein intron through the six montage activity of taking turns the mutant that fallibility PCR obtains.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Embodiment 1

With protein intron Cne PRP8 is that example is carried out orthogenesis.Known Cne PRP8 is not montage in pKH.The encoding sequence that contains protein intron Cne PRP8 among the pMCP.

1) utilize the primer PKHA of a pair of BsaI of containing restriction enzyme site and PKHB that pKH is carried out inverse PCR, after the phosphorylation, connect transformed competence colibacillus certainly, obtain plasmid pKH ', the BsaI enzyme is cut the carrier that just can obtain to be used for orthogenesis.

2) with pMCP be template, utilize primer CPb1 and the CPb2 of a pair of Bsa1 of containing that Cne PRP8 gene is carried out first round fallibility PCR, the amount of pMCP template is 100ng, the BsaI enzyme is cut and is obtained to insert fragment, to insert fragment and 1) in the acquisition carrier be connected with 3: 1 mol ratio, concentrate, transform the two anti-flat board that is coated with the KanR (kantlex) that contains 100 μ g/ml Amp (penbritin) and 20 μ g/ml.

3) above-mentioned two anti-dull and stereotyped bacterium colonies of going up growth are carried out bacterium liquid PCR preliminary evaluation, and obtain the mixing plasmid of positive colony, as the template of next round fallibility PCR.Along with the carrying out of fallibility PCR, the kantlex concentration in two anti-flat boards improves constantly, and so continuous accumulation is of value to the sudden change of protein intron Cne PRP8 montage.Carried out 6 altogether and taken turns fallibility PCR, kantlex concentration is followed successively by K20 μ g/ml, K50 μ g/ml, K100 μ g/ml, K200 μ g/ml, K600 μ g/ml and K700 μ g/ml.

Fig. 4 is classical fallibility PCR picture.Verify that by westernblotting obtaining whether montage efficient of Cne PRP8 mutant obtains to improve, contrast to be plasmid pMCP and pKH that pMCP can produce Cne PRP8, pKH can produce KanR albumen.Fig. 5 for Cne PRP8 through the six montage situations of taking turns the mutant that obtains behind the fallibility PCR, wherein CPE is the protein intron of the montage most effective (78%) that obtains by orthogenesis.

Embodiment 2

2) with pMCP be template, utilize primer CPb1 and the CPb2 of a pair of Bsa1 of containing that Cne PRP8 gene is carried out first round fallibility PCR, the amount of template is 80ng, the BsaI enzyme is cut and is obtained to insert fragment, and the insertion fragment is connected with the mol ratio of carrier with 3: 1, concentrate, transform the two anti-LB flat board that is coated with the KanR (kantlex) that contains 100 μ g/ml Amp (the blue or green benzyl mycin of ammonia) and 20 μ g/ml.

3) bacterium colony of growing on above-mentioned two anti-LB flat boards is carried out bacterium liquid PCR preliminary evaluation, and obtain the mixing plasmid of positive colony, as the template of next round fallibility PCR.Along with the carrying out of fallibility PCR, the kantlex concentration in two anti-flat boards improves constantly, and so continuous accumulation is of value to the sudden change of protein intron Cne PRP8 montage.Carried out 4 altogether and taken turns fallibility PCR, kantlex concentration is followed successively by K20 μ g/ml, K50 μ g/ml, K100 μ g/ml, K200 μ g/ml.The montage efficient of the protein intron that the montage that obtains is most effective is about 56%.

Embodiment 3

2) with pMCP be template, utilize primer CPb1 and the CPb2 of a pair of Bsa1 of containing that Cne PRP8 gene is carried out first round fallibility PCR, the amount of template is 80ng, the BsaI enzyme is cut and is obtained to insert fragment, and the insertion fragment is connected with the mol ratio of carrier with 3: 1, concentrate, transform the two anti-LB flat board that is coated with the KanR (kantlex) that contains 100 μ g/ml Amp (ammonia benzyl mycin) and 20 μ g/ml.

3) bacterium colony of growing on above-mentioned two anti-LB flat boards is carried out bacterium liquid PCR preliminary evaluation, and obtain the mixing plasmid of positive colony, as the template of next round fallibility PCR.Along with the carrying out of fallibility PCR, the kantlex concentration in two anti-flat boards improves constantly, and so continuous accumulation is of value to the sudden change of protein intron Cne PRP8 montage.Carried out 5 altogether and taken turns fallibility PCR, kantlex concentration is followed successively by K20 μ g/ml, K50 μ g/ml, K100 μ g/ml, K200 μ g/ml, K600 μ g/ml.The montage efficient of the protein intron that the montage that obtains is most effective is about 65%.

Claims

1. the method for a fallibility PCR/ kantlex screening system directed evolution of proteins intron comprises:

1) utilizes a pair of primer 5 ' end to add II type restriction enzyme Bsa I carrier pKH is carried out inverse PCR, with inverse PCR product phosphorylation, connect transformed competence colibacillus certainly then, obtain plasmid pKH ', the BsaI enzyme is cut the fragment that 3516bp is reclaimed in the back, promptly obtains to be used for the carrier of orthogenesis;

2) utilize a pair of primer 5 ' end to add II type restriction enzyme Bsa I protein intron is carried out fallibility PCR, be connected with the above-mentioned carrier that is used for orthogenesis then and deactivation, again through concentrating the transformed competence colibacillus cell, be coated with the two anti-flat board that contains ammonia benzyl mycin and kantlex; Wherein fallibility PCR comprises the Mg of basic, normal, high concentration ²⁺, Mn ²⁺And dXTP, totally 12 reactions, the amount of template is 50-100ng among the fallibility PCR;

3) bacterium colony of growing is carried out bacterium liquid PCR checking on above-mentioned two anti-flat boards, and obtain it with the positive clone of preliminary evaluation and mix the template of plasmid as next round fallibility PCR, carry out 4-6 wheel fallibility PCR, the highest protein intron of montage activity that obtains is by the protein intron after the evolution of orthogenesis acquisition.

2. the method for a kind of fallibility PCR/ kantlex screening system directed evolution of proteins intron according to claim 1 is characterized in that: step 2) described in protein intron be Cne PRP8 protein intron.

3. the method for a kind of fallibility PCR/ kantlex screening system directed evolution of proteins intron according to claim 1 is characterized in that: step 2) in fallibility PCR reaction conditions be:

Lower concentration: 10 * amplification buffer, 1 *; 10mM dNTPs, the 250uM/ kind; CPb1,0.5uM; CPb3,0.5uM; 25mM Mg ²⁺, 0.25mM; 5mM Mn ²⁺, 0.05mM; DdH2O, 50ul; Template, 50-100ng; The Tap archaeal dna polymerase, 5U; 10mM d[X] TP, 1000uM;

Intermediate concentration: 10 * amplification buffer, 1 *; 10mM dNTPs, the 250uM/ kind; CPb1,0.5uM; CPb3,0.5uM; 25mM Mg ²⁺, 0.5mM; 5mM Mn ²⁺, 0.1mM; DdH2O, 50ul; Template, 50-100ng; The Tap archaeal dna polymerase, 5U; 10mM d[X] TP, 1500uM;

High density: 10 * amplification buffer, 1 *; 10mM dNTPs, the 250uM/ kind; CPb1,0.5uM; CPb3,0.5uM; 25mM Mg ²⁺, 1.0mM; 5mM Mn ²⁺, 0.2mM; DdH2O, 50ul; Template, 50-100ng; The Tap archaeal dna polymerase, 5U; 10mM d[X] TP, 2000uM.

4. the method for a kind of fallibility PCR/ kantlex screening system directed evolution of proteins intron according to claim 1 is characterized in that: step 2) in insert fragment and carrier when connecting mol ratio be 3: 1.

5. the method for a kind of fallibility PCR/ kantlex screening system directed evolution of proteins intron according to claim 1, it is characterized in that: in the step 3) every take turns fallibility PCR after, improve constantly the concentration of the kantlex in two anti-flat boards, till the colony number on two anti-flat boards no longer increases, and verify its montage efficient with the Western trace.