CN115948439A

CN115948439A - Phage-assisted intein evolution system and method

Info

Publication number: CN115948439A
Application number: CN202211090146.0A
Authority: CN
Inventors: 陈西朋; 张志乾; 罗元廷; 刘丽花; 江翱; 吴奕瑞; 王帆; 吴嵩; 杨敏
Original assignee: Shandong Dry Peptide Biotechnology Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Current assignee: Shandong Dry Peptide Biotechnology Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Priority date: 2022-09-07
Filing date: 2022-09-07
Publication date: 2023-04-11
Anticipated expiration: 2042-09-07
Also published as: CN115948439B

Abstract

The invention discloses a phage-assisted intein evolution system, which comprises: a host bacterium containing the F plasmid; the gIII gene is replaced by a recombinant M13 phage of an N-terminal fusion protein gene, and the N-terminal fusion protein gene is pIII protein N-terminal-intein N-terminal fusion protein; a mutant plasmid for mutating the genome of the M13 bacteriophage; and a gIII helper plasmid for expressing a C-terminal fusion protein, wherein the C-terminal fusion protein is an intein C-terminal-pIII protein C-terminal fusion protein; the intein is a split intein. And a corresponding method of evolution. By using the evolution method, the bacteriophage in the evolution pool is gradually replaced by the filial generation bacteriophage containing the high-activity intein variant DNA sequence in the genome, and the high-activity and low-preference intein can be obtained by separating the filial generation bacteriophage. The method can be used for evolutionarily obtaining the intein variant with high activity and low preference.

Description

Phage-assisted intein evolution system and method

Technical Field

The invention relates to a phage-assisted intein evolution system and method.

Background

An intein is an amino acid sequence located in a protein precursor that catalyzes the self-cleavage of the precursor and is therefore named intein because its mechanism is similar to the splicing of eukaryotic introns. Inteins are similar to proteases and have the activity of cleaving peptide bonds, and therefore, inteins have wide applications in the field of life sciences. Particularly, in protein expression, purification and label excision, the intein has the remarkable advantages of promoting expression, being convenient to cut, being low in cost and the like, so that the intein has important commercial value. However, the inteins still have obvious limitations, such as low catalytic activity and severe preference, which seriously affects the application and popularization of the inteins. Improving the catalytic activity of inteins and reducing the preference of inteins is an urgent problem to be solved.

Directed evolution is a biotechnology emerging in recent years, and it is a protein or RNA engineering technology used to obtain an evolution result in nature for billions of years in months by using a laboratory scale evolution system. The phage-assisted bacterial strain evolution technology, also called PACE technology (phase assisted continuous evolution), is a phage-based enzyme evolution technology developed in 2011 by David r. The core of the technology is to couple the activity of the enzyme with the infection capacity of the phage, namely, the stronger the activity of the enzyme, the higher the titer of the phage. And then, an enzyme mutant is continuously generated in the evolution process by using a random mutagenesis method, and the gene of the enzyme is integrated into the genome of the phage, so that the aim that the phage with higher activity can be packaged by carrying a gene with higher activity on the genome is fulfilled. Finally, the enzyme variant with high activity is screened out by the method. PACE has advantages such as easy operation, low cost and fast evolution speed, so it is widely used in various enzyme evolution fields.

Disclosure of Invention

The invention discloses a phage-assisted intein evolution system, which comprises:

a host bacterium containing the F plasmid;

the gIII gene is replaced by a recombinant M13 phage of an N-terminal fusion protein gene, and the N-terminal fusion protein gene is pIII protein N-terminal-intein N-terminal fusion protein;

a mutant plasmid for mutating the genome of the M13 bacteriophage;

and a gIII helper plasmid for expressing a C-terminal fusion protein, wherein the C-terminal fusion protein is an intein C-terminal-pIII protein C-terminal fusion protein;

the intein is a split intein.

Preferably, one or two of the following conditions are also satisfied:

A. inserting amino acid or oligopeptide changing the cutting preference of the intein between the C end of the intein of the C-end fusion protein and the C end of the pIII protein;

B. and an amino acid or oligopeptide changing the cutting preference of the intein is inserted between the N end of the pIII protein of the N-end fusion protein and the N end of the intein.

Preferably, the intein is Mxe GyrA, ssp DnaE or Mut RecA.

Preferably, the host bacterium is Escherichia coli JM108, JM109, S1030 or S2060.

Preferably, the mutant plasmid is a DP6 plasmid.

Preferably, PPP is inserted between the C terminal of the intein and the C terminal of the pIII protein, and PPP is inserted between the N terminal of the pIII protein and the N terminal of the intein.

Preferably, the sequence of the N-terminal fusion protein gene is shown as SEQ ID N0:2 or SEQ ID N0: 9.

Preferably, the genome sequence of the recombinant M13 phage is shown as SEQ ID N0:3 or SEQ ID N0: 10.

Preferably, the sequence of the C-terminal fusion protein gene is shown as SEQ ID N0:5 or SEQ ID N0: 11.

Preferably, the sequence of the gIII helper plasmid is shown as SEQ ID N0:6 or SEQ ID N0: 12.

Preferably, the sequence of the mutant plasmid is shown as SEQ ID N0:7 or SEQ ID N0: 8.

The invention also discloses a phage-assisted intein evolution method, which is characterized by comprising the following steps: the method comprises the following steps:

(1) Synthesizing an expression gene of pIII protein N-terminal-intein N-terminal fusion protein, and replacing a gIII gene of the M13 bacteriophage with the expression gene of the N-terminal fusion protein to obtain a recombinant M13 bacteriophage;

(2) Synthesizing an expression gene of the intein C-terminal-pIII protein C-terminal fusion protein, and constructing the expression gene into an expression vector to obtain a gIII auxiliary plasmid;

(3) Synthesizing a mutant plasmid;

(4) Preparation of evolved strains: transferring gIII auxiliary plasmid and mutant plasmid into colibacillus strain, screening positive clone as evolved strain;

(5) And (3) phage infection evolution: infecting the evolved bacterial strain with recombinant M13 bacteriophage, continuously mutating bacteriophage genome with mutation plasmid, promoting auxiliary plasmid to express more pIII protein with high activity intein variant, finally packaging the DNA sequence corresponding to the intein variant into high activity new generation bacteriophage, and further proceeding next infection and mutation evolution. By using the method, the bacteriophage in the evolution pool is gradually replaced by the filial bacteriophage containing the DNA sequence of the high-activity intein variant in the genome, and the high-activity and low-preference intein can be obtained by separating the filial bacteriophage.

The invention has the beneficial effects that:

the invention discloses a phage-assisted intein evolution method, which comprises a host strain containing F plasmid, an M13 phage (gIII gene deletion) containing intein sequence in genome, a mutant plasmid and a gIII helper plasmid containing response intein activity. The mutant plasmid and the gIII helper plasmid are jointly transformed into a host strain, M13 phage is used for infecting the host strain, the mutant plasmid continuously mutates a phage genome, the high-activity intein variant promotes the helper plasmid to express more pIII proteins, finally, a DNA sequence corresponding to the intein variant is packaged into a new generation of phage with high activity, and then, the next step of infection and mutation evolution is carried out. By using the method, the bacteriophage in the evolutionary pool is gradually replaced by the filial generation bacteriophage containing the DNA sequence of the high-activity intein variant in the genome, and the high-activity and low-preference intein can be obtained by separating the filial generation bacteriophage. The method can be used for evolutionarily obtaining the intein variant with high activity and low preference.

Drawings

FIG. 1 is a graph of the complete pIII expression plotted against the intein activity pair.

FIG. 2 is a graph of the complete pIII expression vs. intein vs. activity coupling at different amino acid termini.

FIG. 3 is a schematic diagram of the genome of an intein-evolving phage M13 in which the gIII gene position of the wild-type M13 phage genome has been modified.

FIG. 4 progeny phage assembly without helper plasmid.

FIG. 5 schematic representation of helper plasmid engineering.

FIG. 6 progeny phage assembly in the presence of helper plasmid and intein activity.

FIG. 7 schematic representation of the mutant plasmid.

FIG. 8 schematic diagram of evolved strains.

FIG. 9 is a schematic representation of the primary phage preparation.

FIG. 10 is a graph of the evolution pattern of a low-activity phage.

FIG. 11 is a diagram showing the evolution pattern of a highly active phage.

FIG. 12 is a diagram of an evolutionary equilibrium pattern.

FIG. 13 three-dimensional structure diagram of Mxe GyrA intein.

FIG. 14 schematic representation of N-and C-terminal structures after resolution of Mxe GyrA intein.

FIG. 15M 13 phage genome map.

FIG. 16 engineered M13 phage genome map.

Figure 17pJC175e plasmid map.

FIG. 18 map of the engineered helper plasmid.

FIG. 19DP6 plasmid map.

FIG. 20 engineered mutant plasmid map.

FIG. 21Western blot verifies the efficiency of intein cleavage before and after evolution.

FIG. 22 comparison of intein cleavage efficiency before and after evolution.

Figure 23 engineered M13 phage genome (proline) map.

FIG. 24 modified helper plasmid (proline) profile.

FIG. 25Western blot to verify the efficiency of intein (proline) cleavage before and after evolution.

FIG. 26 comparison of intein (proline) cleavage efficiency before and after evolution.

Detailed Description

The invention is further illustrated below by the scheme of the phage-assisted intein evolution process, and these specific examples should not be construed in any way as limiting the scope of application of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 design of phage-assisted intein evolution method.

This example designed a phage-assisted intein evolution approach.

Design of intein activity coupling pIII protein activity. The Intein can be split into two parts of N end and C end, namely split Intein (split Intein). The split inteins can splice together proteins linked at both ends by trans-splicing. Such as Mxe GyrA, ssp DnaE, mut RecA, etc. The pIII protein can also be split into an N end and a C end, the N end is a signal peptide, the C end is an active part, and only the N end and the C end are fused into the complete pIII protein, so that the phage with the infection activity can be packaged. The N end of the pIII protein is fused with the N end of the intein to form a fusion protein, and the C end of the intein is fused with the C end of the pIII protein to form the fusion protein. The more active the intein, the more active pIII protein is produced. The mechanism of action of intein-mediated trans-splicing of pIII is shown in figure 1. Different amino acids or oligopeptides are inserted between the inteins and gIII, i.e.inteins of different preference can be used for evolution. The mechanism of action is shown in FIG. 2.

M13 phage genome design of the evolved intein. Based on the principle of intein activity coupling pIII protein activity in fig. 1 and 2, we designed the M13 phage genome. On the M13 phage genome, we retained the N-terminal portion of the gIII gene (the first 10 amino acids), while the C-terminus of the gIII gene was replaced by the N-terminus of the intein (see FIGS. 1, 2 and 3). Thus, the designed M13 phage was unable to infect the host bacteria because it was unable to independently express the active complete gIII protein (see FIG. 4).

Designing helper plasmid containing gIII. Based on the principle of intein activity coupled pIII protein activity in FIG. 2, we designed a gIII plasmid design to aid phage assembly. The fusion protein of the C-terminal of the intein and the C-terminal of the pIII protein is induced and regulated to express on a phage inducible expression promoter (PSPO) (see figures 2 and 5). Only when M13 phage infects host bacteria, pIV protein expressed by gIV gene of phage starts PSPO expression, and can translate into fusion protein containing peptide C end and pIII protein C end (see figure 6).

Design of mutant plasmids. Referring to FIG. 7, the mutant plasmid contains 6 mutagenized genes, i.e., low fidelity DNA polymerase dnaQ926, error prone DNA polymerase polV, DNA methylase dam, DNA deaminase cda1, DNA repair inhibitory protein ugi and mutation maintenance protein emrR. In addition, three tetO operons were added after the PSPO promoter, together controlling the expression of the complete gIII gene.

Example 2 phage helper intein evolution protocol

This example discloses a phage-assisted intein evolution procedure:

and (4) preparing the evolved strain. And S1030, transferring the escherichia coli strain into a gIII helper plasmid and a mutant plasmid, carrying out overnight culture in a glucose-containing ampicillin and chloramphenicol double-resistant LB solid culture medium, and selecting a monoclonal antibody to be cultured in a glucose-containing ampicillin and chloramphenicol double-resistant LB liquid culture medium until the OD value is about 0.8. The evolved strain was stored at-80 ℃ and revived once a week for use. See fig. 8.

And (3) preparing primary phage. Transferring the constructed M13 genome plasmid into an evolved strain, adding tetracycline for induction, separating M13 phage plaques by using a double-layer agarose plate method, picking a single phage into the evolved strain, and performing sequencing verification after culture. See fig. 9.

And (5) evolution of low-activity phage. The obtained phages were mixed according to 1:1000 infecting host bacteria, when the activity of the intein variant is lower, the generated pIII protein is less, and the assembled M13 bacteriophage with infection activity is less. Expression of the mutagenized gene in the mutant plasmid was induced by the addition of arabinose to produce intein mutants. After a period of time, tetracycline is added to induce the expression of normal gIII, so as to obtain high-efficiency progeny phage. See fig. 10.

And (5) high-activity phage evolution. The obtained phages were mixed according to 1:1000 infect host bacteria, when the activity of the intein variant is higher, more pIII protein is generated, and more M13 bacteriophage with infection activity is assembled. Expression of the mutagenized gene in the mutant plasmid was induced by the addition of arabinose to produce intein mutants. After the mutated phage genome with high activity intein infects host bacteria, more phage can be assembled, and the phage occupies a proportional advantage in an evolution pool. After mutation, the phage genome with low activity intein is infected by host bacteria, so that fewer phage can be assembled and the proportion of phage in the evolution pool is low. Thereby realizing the directed evolution of the high-activity intein. See fig. 11.

And (5) carrying out evolutionary balance. When the intein activity reaches a certain threshold, it is uncoupled from the expression of intact pIII. That is, the activity change of the intein can not greatly affect the expression of pIII, when the titer of the phage in the evolution pool can reach the titer threshold value in a short time, the evolution enters a balance stage, and the evolution is finished. See fig. 12.

Example 3 evolution of high Activity Mxe GyrA inteins

This example discloses the evolution of high activity Mxe GyrA inteins.

(1) Resolution of Mxe GyrA intein. The amino acid sequence of Mxe GyrA is:

<xnotran> CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHN (SEQ ID No. 13), SWISS-MODEL . </xnotran> As a result, as shown in FIG. 13, mxe GyrA can be cleaved from the 119 th to 120 th amino acids to produce two isolated polypeptides. The N terminal comprises 119 amino acids with the sequence of

<xnotran> CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFAR (SEQ ID No. 14); </xnotran> The C terminal comprises 79 amino acids with the sequence of

<xnotran> GKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHN (SEQ ID No. 15). </xnotran> The results are shown in FIG. 14.

(2) And (3) splitting the pIII protein. The amino acid sequence of the pIII protein is shown in

<xnotran> MKKLLFAIPLVVPFYSHSAETVHHHHHHAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES (SEQ ID No. 16). </xnotran> pIII which can be cleaved from amino acids 28-29, yields two isolated polypeptides. The N-terminal comprises 28 amino acids and has the sequence MKKLLFAIPLVVPFYSHSAETVHHHHHHHHHH (SEQ ID No. 17); the C end comprises 406 amino acids with the sequence of

AETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES(SEQ ID No.18)。

(3) Mxe GyrA intein activity was co-coupled with intact pIII protein expression. The N end of the pIII protein is coupled with the N end of Mxe GyrA intein to form an N-end fusion protein with the sequence as

<xnotran> MKKLLFAIPLVVPFYSHSAETVHHHHHHCITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHN (SEQ ID No. 19). </xnotran> The C end of Mxe GyrA intein is coupled with the C end of pIII protein to form C-end fusion protein with the sequence as

<xnotran> MGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHNAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES (SEQ ID No. 20). </xnotran> When Mxe GyrA functions as an intein, the C-terminus and N-terminus of the pIII protein are fused to form the complete pIII protein, as shown in FIG. 1.

(4) And (5) constructing a recombinant phage genome. The M13 phage complete genome DNA sequence is shown in SEQ ID No.1, and the map is shown in FIG. 15. The DNA sequence of the N-terminal fusion protein (SEQ ID No. 2) replaces the DNA sequence of the gIII gene, the DNA sequence of the M13 bacteriophage complete genome after recombination is shown in SEQ ID No.3, and the map is shown in FIG. 16. Gene synthesis was performed by Kinry Biotechnology, inc.

(5) Constructing pIII protein activity auxiliary plasmid. The DNA sequence of pJC175e plasmid is shown in SEQ ID No.4, and the map is shown in FIG. 17. The DNA sequence of the C-terminal fusion protein (SEQ ID No. 5) replaces the DNA sequences of the gIII, luxA and LuxB genes, the DNA sequence of the recombinant helper plasmid is shown in SEQ ID No.6, and the map is shown in FIG. 18. Gene synthesis was performed by Kinry Biotechnology, inc.

(6) And (5) constructing a mutant plasmid. The DNA sequence of the DP6 plasmid is shown in SEQ ID No.7, and the map is shown in FIG. 19. The tetO fragment was inserted 3 times in duplicate, and the DNA sequence of the repeat mutant plasmid is shown in SEQ ID No.8, and the map is shown in FIG. 20. Gene synthesis was performed by Kinry Biotechnology, inc.

(7) And (4) preparing an evolved strain. 1030 Escherichia coli strain is transferred into gIII helper plasmid and mutant plasmid, and cultured overnight in 100mg/L ampicillin and 50mg/L chloramphenicol double-resistant LB solid medium containing 10g/L glucose, and monocloned is picked up and cultured in 100mg/L ampicillin and 50mg/L chloramphenicol double-resistant LB liquid medium containing 10g/L glucose until OD value is about 0.8. The evolved strain was stored at-80 ℃.

(8) And (3) preparing primary phage. Transferring the constructed M13 genome plasmid into an evolved strain, adding 1mg/L tetracycline for induction for 2h, separating M13 phage plaques by using a double-layer agarose plate method, picking a single phage into the evolved strain, culturing for 4h, and then carrying out sequencing verification.

(9) And (5) evolution of low-activity phage. The obtained phages were mixed according to 1:1000 infecting host bacteria, incubating at 37 deg.C for 10min, and adding 10mg/L arabinose to induce the expression of mutagen in mutant plasmid to generate mutant of intein. Induction was carried out at 37 ℃ for 6h. Detecting the titer of the phage when the titer is less than 10 ⁶ CFU/ml, adding 1mg/L tetracycline to induce the expression of normal gIII, and obtaining high-efficiency progeny phage. Until the titer of the phage is higher than 10 ⁸ CFU/ml. And (4) centrifuging at 8000rpm for 5min at 4 ℃, collecting the progeny phage supernatant, and entering the next round of evolution.

(10) And (5) high-activity phage evolution. The obtained phages were mixed according to 1:1000 infecting host bacteria, incubating at 37 deg.C for 10min, adding 10mg/L arabinose to induce the expression of mutagen in mutant plasmid, generating mutant of intein. Induction was carried out at 37 ℃ for 6h. Detecting the titer of the phage when the titer is higher than 10 ⁶ CFU/ml,8000rpm 4 ℃ centrifugation for 5min, collecting progeny phage supernatant, and entering the next round of evolution.

(11) And (5) carrying out evolutionary balance. The obtained phages were mixed according to 1:1000 infecting host bacteria, incubating at 37 deg.C for 10min, and adding 10mg/L arabinose to induce the expression of mutagen in mutant plasmid to generate mutant of intein. Induction was carried out at 37 ℃ for 6h. And (4) detecting the titer of the phage, centrifuging for 5min at 8000rpm and 4 ℃ when the titer is higher than 108CFU/ml, collecting the supernatant of the progeny phage, and terminating evolution.

(12) And (4) detecting the activity of the intein after evolution. Equal amounts of the phage before and after evolution were taken and the ratio of 1:10, infecting host bacteria, and incubating for 1h at 37 ℃. The samples were subjected to Western blot detection using His-tagged antibodies, and the results are shown in FIG. 21. The Mxe GyrA intein variants in the evolved phage produced more of the intact pIII protein, about 5-fold more active than the undeveloped Mxe GyrA, see figure 22.

Example 4 evolution of Low proline inhibiting preferential Mxe GyrA inteins

Traditional inteins have significant amino acid preference, especially the cleavage site adjacent to proline, which significantly inhibits the activity of the inteins. In this example, we published an evolutionary scheme for low proline inhibitory bias Mxe GyrA inteins.

(1) The activity of Mxe GyrA inteins on proline adjacent to the cleavage site was co-coupled with the expression of the complete pIII protein. The N end of the pIII protein is coupled with the N end of Mxe GyrA intein, three prolines are used for connection in the middle to form an N-end fusion protein, and the sequence is

<xnotran> MKKLLFAIPLVVPFYSHSAETVHHHHHHPPPCITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHN (SEQ ID No. 21). </xnotran> The C end of Mxe GyrA intein is coupled with the C end of pIII protein, three prolines are used for connection in the middle to form N-end fusion protein, and the sequence is

<xnotran> MGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHNPPPAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES (SEQ ID No. 22). </xnotran> When Mxe GyrA functions as an intein, the C-and N-termini of the pIII protein are fused to form the complete pIII protein (6 prolines in the middle), as shown in FIG. 2.

(2) And (5) constructing a recombinant phage genome. The DNA sequence of the N-terminal fusion protein (SEQ ID No. 9) replaces the DNA sequence of the gIII gene, the DNA sequence of the M13 bacteriophage complete genome after recombination is shown in SEQ ID No.10, and the map is shown in FIG. 23. Gene synthesis was performed by Kinry Biotechnology, inc.

(3) Constructing pIII protein activity auxiliary plasmid. The DNA sequence of the C-terminal fusion protein (SEQ ID No. 11) is used for replacing the DNA sequences of the gIII, luxA and LuxB genes, the DNA sequence of the recombinant helper plasmid is shown in SEQ ID No.12, and the map is shown in FIG. 24. Gene synthesis was performed by Kinry Biotechnology, inc.

(4) And (5) constructing a mutant plasmid. The DNA sequence of the repeated mutant plasmid is shown in SEQ ID No.8, and the map is shown in FIG. 20. Gene synthesis was performed by Kinry Biotechnology, inc.

(5) And (4) preparing the evolved strain. 1030 Escherichia coli strain is transferred into gIII helper plasmid and mutant plasmid, and cultured overnight in 100mg/L ampicillin and 50mg/L chloramphenicol double-resistant LB solid medium containing 10g/L glucose, and monocloned is picked up and cultured in 100mg/L ampicillin and 50mg/L chloramphenicol double-resistant LB liquid medium containing 10g/L glucose until OD value is about 0.8. The evolved strain was stored at-80 ℃.

(6) And (3) preparing primary phage. And transferring the constructed M13 genome plasmid into an evolved strain, adding 1mg/L tetracycline for induction for 2h, separating M13 phage plaques by using a double-layer agarose plate method, picking a single phage into the evolved strain, culturing for 4h, and then performing sequencing verification.

(7) And (5) evolution of low-activity phage. The obtained phages were mixed according to 1:1000 infecting host bacteria, incubating at 37 ℃ for 10min, inducing the expression of the mutagenized gene in the mutant plasmid by adding 10mg/L arabinose, and generating the mutant of the intein. Induction was carried out at 37 ℃ for 6h. Detecting the titer of the phage, when the titer is less than 10 ⁶ CFU/ml, adding 1mg/L tetracycline to induce the expression of normal gIII, and obtaining high-efficiency progeny phage. Until the phage titer is higher than 10 ⁸ CFU/ml. And (4) centrifuging at 8000rpm for 5min at 4 ℃, collecting the progeny phage supernatant, and entering the next round of evolution.

(8) And (5) high-activity phage evolution. The obtained phages were mixed according to 1:1000 infecting host bacteria, incubating at 37 ℃ for 10min, inducing the expression of the mutagenized gene in the mutant plasmid by adding 10mg/L arabinose, and generating the mutant of the intein. Induction was carried out at 37 ℃ for 6h. Detecting the titer of the phage when the titer is higher than 10 ⁶ CFU/ml,8000rpm 4 ℃ centrifugation for 5min, collecting progeny phage supernatant, and entering the next round of evolution.

(9) And (5) carrying out evolutionary balance. The obtained phages were mixed according to 1:1000 infecting host bacteria, incubating at 37 ℃ for 10min, inducing the expression of the mutagenized gene in the mutant plasmid by adding 10mg/L arabinose, and generating the mutant of the intein. Induction was carried out at 37 ℃ for 6h. Detecting the titer of the phage when the titer is higher than 10 ⁸ CFU/ml,8000rpm 4 ℃ centrifugation for 5min, collecting progeny phage supernatant, evolution termination.

(10) And detecting the activity of the intein after evolution. Equal amounts of pre-and post-evolutionary phage were taken and the ratio was adjusted according to 1:10, and incubating for 1h at 37 ℃. The samples were subjected to Western blot detection using His-tagged antibodies, and the results are shown in FIG. 25. The Mxe GyrA intein variants in the evolved phage produced more intact pIII proteins, about 30-fold more active than the non-evolved Mxe GyrA, see fig. 26.

Claims

1. A phage-assisted intein evolution system comprising:

a host bacterium containing the F plasmid;

a mutant plasmid for mutating the genome of the M13 bacteriophage;

the intein is a split intein.

2. The phage intein evolution system according to claim 1, characterized in that one or two of the following conditions are also fulfilled:

3. The phage intein evolution system according to claim 1 or 2, characterized in that: the inteins are MxeGyrA, ssp DnaE or Mut RecA.

4. The phage intein evolution system according to claim 1 or 2, characterized in that: the host bacterium is Escherichia coli JM108, JM109, S1030 or S2060.

5. The phage intein evolution system according to claim 1 or 2, characterized in that: the mutant plasmid is the DP6 plasmid.

6. The phage intein evolution system of claim 2, characterized in that: PPP is inserted between the C end of the intein and the C end of the pIII protein, and PPP is inserted between the N end of the pIII protein and the N end of the intein.

7. The phage intein evolution system of claim 1 or 2, characterized in that: the sequence of the N-terminal fusion protein gene is shown as SEQ ID N0:2 or SEQ ID N0: 9.

8. The phage intein evolution system according to claim 1 or 2, characterized in that: the genome sequence of the recombinant M13 phage is shown as SEQ ID N0:3 or SEQ ID N0: 10.

9. The phage intein evolution system according to claim 1 or 2, characterized in that: the sequence of the C-terminal fusion protein gene is shown as SEQ ID N0:5 or SEQ ID N0: 11.

10. The phage intein evolution system of claim 1 or 2, characterized in that: the sequence of the gIII helper plasmid is shown as SEQ ID N0:6 or SEQ ID N0: 12.

11. The phage intein evolution system of claim 5, characterized in that: the sequence of the mutant plasmid is shown as SEQ ID N0:7 or SEQ ID N0: 8.

12. A method for phage-assisted intein evolution, characterized in that: the method comprises the following steps:

(3) Synthesizing a mutant plasmid;

(5) And (3) phage infection evolution: infecting the evolved strain with recombinant M13 phage to obtain the evolved intein.