CN115896147B - Intein evolution systems and methods, corresponding mutant plasmids and reporter plasmids - Google Patents

Abstract

The invention discloses an intein evolution system, which comprises the following steps: a mutant plasmid comprising an expressed gene of a nicking enzyme and a low fidelity DNA polymerase, and an expressed gene of a dimerized short peptide system consisting of two short peptides; a reporter plasmid comprising a fusion protein expression gene of an antibiotic resistance protein-N and an intein-N gene, and a fusion protein expression gene of an intein-C and an antibiotic resistance protein-C gene; and expression vectors for the mutant plasmids and reporter plasmids. The mutant plasmids and reporter plasmids described above, as well as corresponding methods of intein evolution, are also disclosed. The invention adopts the incision enzyme and the DNA polymerase to continuously mutate the intein gene, and the intein gene with enhanced activity generates more complete antibiotic resistance proteins through the splicing action of the intein, so that the strain obtains high antibiotic resistance. And reading through high-resistance culture screening to obtain a high-resistance strain, wherein the strain is a strain containing high-activity intein.

Description

Intein evolution systems and methods, corresponding mutant plasmids and reporter plasmids

Technical Field

The invention relates to an intein evolution system and method, corresponding mutant plasmids and reporter plasmids.

Background

Inteins are amino acid sequences located in protein precursors that catalyze cleavage of themselves from the precursor, and are therefore termed inteins because their mechanism resembles splicing of eukaryotic introns. Inteins are similar to proteases and have the activity of cleaving peptide bonds. Thus, inteins are widely used in the field of life sciences. Particularly in protein expression, purification and tag excision, inteins have significant advantages such as promotion of expression, convenient cleavage and low cost, and thus, inteins have important commercial value. However, inteins still have significant limitations, such as low catalytic activity and severe preference, which severely affects the use and popularization of inteins. Increasing the catalytic activity of inteins and decreasing the preference of inteins is a highly desirable problem.

Directed evolution is a biological technology which has been raised in recent years, and by utilizing a laboratory-scale evolution system, the evolution result which needs billions years in nature can be obtained in a period of months, and the directed evolution is a protein or RNA modification technology. How to obtain high-activity inteins by directed evolution technology is a focus of industry.

Disclosure of Invention

The invention discloses a mutant plasmid, which comprises expression genes of a nicking enzyme and a low-fidelity DNA polymerase and expression genes of a dimerization short peptide system consisting of two short peptides, wherein one short peptide in the dimerization short peptide system and the nicking enzyme form fusion proteins, the short peptide is positioned at the N end or the C end of the nicking enzyme, the other short peptide in the dimerization short peptide and the low-fidelity DNA polymerase form fusion proteins, and the short peptide is positioned at the N end or the C end of the low-fidelity DNA polymerase.

Preferably, the sequence of the plasmid is shown as SEQ ID No. 1.

The invention also discloses a report plasmid, which is characterized in that: the reporter plasmid contains a fusion protein expression gene of the antibiotic resistance protein-N and the intein-N gene and a fusion protein expression gene of the intein-C and the antibiotic resistance protein-C gene.

Preferably, the reporter plasmid further comprises a nicking enzyme recognition site, and the nicking enzyme recognition site is disposed upstream or downstream of each fusion expressed gene.

Preferably, the sequence of the reporter plasmid is shown in SEQ ID No. 2.

The invention also discloses an intein evolution system, which is characterized by comprising the following steps:

the mutant plasmids described above;

the above-mentioned reporter plasmid;

expression vectors for mutant plasmids and reporter plasmids.

Preferably, the expression vector is E.coli.

The invention also discloses an intein evolution method, which is characterized by comprising the following steps:

(1) Obtaining the mutant plasmid and the report plasmid;

(2) Transforming the mutant plasmid and the reporter plasmid into an expression vector, and screening out an expression vector monoclonal which is transformed with the mutant plasmid and the reporter plasmid simultaneously;

(3) And (3) adding antibiotic resistance protein of a reporter plasmid, expressing the reporter plasmid, simultaneously inducing the mutant plasmid to express, and culturing and screening the single clone of the expression vector screened in the step (2) under a high-resistance environment to obtain the expression vector containing the intein after evolution.

Preferably, the antibiotic resistance protein is kanamycin resistance protein.

Preferably, the mutant plasmid expression is induced with arabinose.

The invention has the beneficial effects that:

the invention discloses an intein evolution system which comprises an evolution strain, a mutant plasmid and a reporter plasmid regulated and controlled by intein activity. The principle of evolution is that a nicking enzyme and a low fidelity DNA polymerase are assembled together through dimerization of short peptides, the nicking enzyme and the DNA polymerase continuously mutate intein genes, and the intein genes with enhanced activity generate more complete antibiotic resistance proteins through splicing action of the intein, so that the strain obtains high antibiotic resistance. And reading through high-resistance culture screening to obtain a high-resistance strain, wherein the strain is a strain containing high-activity intein. By the intein evolution system, strains containing high-activity inteins can be rapidly screened and obtained.

Drawings

FIG. 1 is a schematic diagram of a mutant plasmid.

FIG. 2 is a schematic diagram of an intein active coupling reporter plasmid.

FIG. 3 relationship between intein variant activity and Kana resistance.

FIG. 4 number of clones under different concentrations of Kana resistant plating conditions.

FIG. 5 expression level of intact KanaR protein under the conditions of different concentrations of Kana resistant liquid medium.

Detailed Description

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

Example 1

The mutant plasmid is synthesized according to the sequence of SEQ NO.1, the report plasmid is synthesized according to the sequence of SEQ NO.2, 100ng of the report plasmid and 100ng of the mutant plasmid are transferred into ice bath in the competence of escherichia coli DH5a for 30min, and the temperature is 42 ℃ for 45s, and the ice bath is immediately carried out for 5min. LB medium was added and left to stand overnight at room temperature.

200ul of bacterial liquid is taken and coated in LB solid medium containing 30mg/L of ampicillin and 50mg/L of chloramphenicol, and the mixture is kept stand for 30min. The plates were inverted and incubated overnight in a 37℃incubator.

The monoclonal transformed with mutant plasmid and reporter plasmid is picked up and cultured in LB solid medium containing 500mg/L ampicillin and 50mg/L chloramphenicol at 220rpm and 37 ℃ until OD600 reaches 0.3-0.4.

0.5mg/L tetracycline was added and incubation continued for 4h at 37℃at 220 rpm. The strain was washed 3 times with fresh LB after centrifugation at 4000rpm for 10 min.

The strain was suspended in fresh LB medium, and the strain was subjected to gradient dilution using fresh LB medium, and 50ul of the suspended strain was spread on LB solid medium containing 0, 10, 20, 40, 80, 160mg/L kanamycin, and allowed to stand for 30min. The plates were inverted and incubated overnight in a 37℃incubator.

And counting monoclonal colonies on the solid plate, and multiplying the monoclonal colonies by a gradient dilution multiple to obtain the clone number of the strain containing the concentration of the kana resistance.

The structure of the mutant plasmid is shown in figure 1, the structure of the reporter plasmid regulated by the activity of the intein is shown in figure 2, and the principle of the intein evolution method is shown in figure 3.

The results of the number of clones of the kanamycin-resistant strain before and after mutation are shown in FIG. 4. The number of clones of the strain containing kanamycin resistance after mutation was significantly increased, which suggests that intein mutation improved the ability to produce the complete kanamycin resistance gene, i.e., the activity of the intein was enhanced.

Example 2

1ul of the strain after mutagenesis in example 1 was added to 100ml of LB liquid medium containing 0, 10, 20, 40, 80, 160mg/L kanamycin, 220rpm, and incubated at 37℃until OD600 reached 0.6-0.7.

40ul of the cultured bacterial liquid is taken, 10ul of 5 XSDS-PAGE loading buffer is added, and the mixture is uniformly mixed and then is subjected to boiling water bath for 10-20min. Centrifuging at 12000rpm for 1min, and collecting supernatant.

10ul of the supernatant was spotted into 12% SDS-PAGE gels and electrophoresed at 120V for 1h.

Proteins were transferred to PVDF membrane using an electrotransfer apparatus, and electrophoresis was performed for 2h at 220 mA.

The PVDF membrane was incubated with 5% BSA solution overnight, diluted 1:10000 with Abclonal HRP-conjugated Mouse anti His-Tag mAb (AE 028) for 2h, and washed 4 times 5min each with PBST. Development was performed using ECL limited to the kit and photographing was performed on an imager.

The results are shown in FIG. 5, where the evolved inteins were able to produce more KanaR complete protein.

Claims (6)

1. The application of the mutant plasmid in intein evolution, wherein the sequence of the mutant plasmid is shown as SEQ ID No. 1.

2. An intein evolution system, comprising:

a mutant plasmid shown in SEQ ID No. 1;

a reporter plasmid shown in SEQ ID No. 2;

expression vectors for mutant plasmids and reporter plasmids.

3. The intein evolution system of claim 2, wherein: the expression vector is escherichia coli.

4. An intein evolution method characterized by the steps of:

(1) Obtaining a mutant plasmid shown as SEQ ID No.1 and a reporter plasmid shown as SEQ ID No. 2;

(2) Transforming the mutant plasmid and the reporter plasmid into an expression vector, and screening out an expression vector monoclonal which is transformed with the mutant plasmid and the reporter plasmid simultaneously;

(3) And (3) adding antibiotic resistance protein of a reporter plasmid, expressing the reporter plasmid, simultaneously inducing the mutant plasmid to express, and culturing and screening the single clone of the expression vector screened in the step (2) under a high-resistance environment to obtain the expression vector containing the intein after evolution.

5. The intein evolution method of claim 4, wherein: the antibiotic is kanamycin.

6. The intein evolution method according to claim 4 or 5, wherein: mutant plasmid expression was induced with arabinose.