CN101509028A

CN101509028A - Method for orthogenesis with differ-degree continuous-fallibility PCR

Info

Publication number: CN101509028A
Application number: CNA2009100480205A
Authority: CN
Inventors: 孟清; 周倩
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2009-03-23
Filing date: 2009-03-23
Publication date: 2009-08-19

Abstract

The invention relates to a method for directional evolution using continuous error-prone PCR of different degrees, comprising: selecting a target gene as a template to design primers; preparing d[X]TP error-prone PCR systems with different degrees of high, medium and low; A good PCR reaction system is put into a PCR machine for PCR amplification; the error-prone PCR product is recovered by agarose gel electrophoresis, digested, connected with a carrier, transformed, positive clones are screened, and the plasmid is extracted; the extracted plasmid As a template, the second round of error-prone PCR is carried out; the corresponding screening system is used to screen for useful mutations. The method is simple to operate, has high mutation frequency and stable yield, and can provide new ideas and approaches for directed evolution of proteins.

Description

A kind of method of utilizing continuous error-prone PCR of different degrees to carry out orthogenesis

Technical field

The present invention relates to biotechnology protein engineering research field, relate in particular to a kind of method of utilizing continuous error-prone PCR of different degrees to carry out orthogenesis.

Background technology

Orthogenesis (Directed evolution) is the new technology that developed recently gets up, and is extension and the application of Darwinian evolutionsim thought on nucleic acid, peptide or albumen equimolecular level.It does not need to understand in depth proteinic structure-function relationship, manual simulation biomacromolecule nature evolutionary process under laboratory condition, external gene is carried out random mutagenesis, make gene that a large amount of variations take place, and orthoselection goes out the mutant of required character, thereby can realize the evolutionary process that nature just can be finished in millions of years at short notice.

Orthogenesis technology utilization two kinds of technology the most widely is fallibility PCR (error prone PCR) and DNA reorganization (DNAshuffling), its core concept is the gene order that changes coded protein randomly, obtain the library, from the library, obtain the goal gene that forward is evolved by a large amount of screenings then.Wherein fallibility PCR is a kind of easy method of making sudden change apace in dna sequence dna at random, and it is by changing some component concentrations in the conventional P CR reaction system, makes base mispairing and introduce multipoint mutation at random to a certain extent.The key of this kind method is to select suitable mutation frequency, when mutation frequency is too high, almost can't screen useful sudden change; If mutation frequency is too low, wild-type will occupy the advantage of mutagenized populations, also be difficult to screen the ideal mutant.In general, suitable mutation frequency is 2～3 bases of each sequence or 1 amino-acid residue sudden change.Often be difficult to obtain satisfied result because one takes turns fallibility PCR, continuous error-prone PCR method therefore commonly used, the favourable mutator gene that pcr amplification is obtained is as the template of amplification next time, carry out random mutagenesis continuously repeatedly, make each time the micromutation accumulation that obtains and produce important useful sudden change.The present invention has introduced high, medium and low three kinds of fallibility PCR simultaneous mutations in various degree, with the screening sudden change storehouse that reaches specific mutation frequency and obtain some amount.

Summary of the invention

Technical problem to be solved by this invention provides a kind of method of utilizing high, normal, basic continuous error-prone PCR of different degrees rapidly and efficiently to carry out orthogenesis, and this method is by adding the Mg of various dose ²⁺, Mn ²⁺, and the interpolation concentration ratio of dATP, dTTP, dCTP, dGTP base in the change system, increase the mispairing rate, simple to operate, mutation frequency height, stable yield can be directed evolution of proteins new thinking and approach are provided.

The method of utilizing continuous error-prone PCR of different degrees to carry out orthogenesis provided by the present invention comprises the steps:

(7) selecting goal gene is template design primer;

(8) the high, medium and low d[X in various degree of preparation] the TP error-prone PCR systems;

(9) prepared PCR reaction system is put into the PCR instrument and carried out pcr amplification;

(10) fallibility PCR product is reclaimed by agarose gel electrophoresis, carry out enzyme and cut, be connected, transform, carry out the positive colony screening, extract plasmid with carrier;

(11) be template with the plasmid that extracts, repeating step (1)～(4) are carried out second and are taken turns fallibility PCR;

(12) repeating step (5) utilizes corresponding screening system to carry out the screening of useful sudden change.

In the error-prone PCR systems of the described low degree such as sudden change such as grade of step (2): 25mmol/L Mg ²⁺Final concentration be 0.25mmol/L, 5mmol/L Mn ²⁺Final concentration be 0.05mmol/L, 10mmol/L d[X] final concentration of TP is 1000 μ mol/L.

In the error-prone PCR systems of the described medium sudden change degree of step (2): 25mmol/L Mg ²⁺Final concentration be 0.5mmol/L, 5mmol/LMn ²⁺Final concentration be 0.1mmol/L, 10mmol/L d[X] final concentration of TP is 1500 μ mol/L.

In the error-prone PCR systems of the described high sudden change degree of step (2): 25mmol/L Mg ²⁺Final concentration be 1.0mmol/L, 5mmol/L Mn ²⁺Final concentration be 0.2mmol/L, 10mmol/L d[X] final concentration of TP is 2000 μ mol/L.

In the described PCR system of step (2), used polysaccharase is the Taq enzyme.

The described PCR reaction conditions of step (3) is: 94 ℃ of sex change in 3 minutes and enzyme activate, 94 ℃ of sex change in 45 seconds, and 58 ℃ of annealing in 30 seconds 30 seconds, 72 ℃ were extended in 90 seconds, circulated 35 times; Last 72 ℃ of extensions in 10 minutes.

TBE buffer is used in the described agarose gel electrophoresis analysis of step (4), and gum concentration is 1.0%, and gel reclaims test kit and uses Qiagen company product.

The present invention utilizes high, normal, basic three kinds of methods of continuous error-prone PCR in various degree, can effectively control mutation frequency, obtains a certain size sudden change storehouse.The present invention is simple to operate, the mutation frequency height, and stable yield, controllability is strong, in conjunction with corresponding screening means, for the orthogenesis engineered protein provides new thinking and approach.

Description of drawings

The fallibility PCR agarose gel electrophoresis result of Fig. 1 embodiment 1, M is marker, the positive contrast of P;

The western blot of Fig. 2 embodiment 2 detects mutant montage activity,

1 negative contrast, 2 positive contrasts, 3～9 is the mutein intron of preliminary screening.

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: the orthogenesis of protein intron

1. select the protein intron Ssp DnaB of montage active about 30% to carry out fallibility PCR, the design primer is as follows:

5’-AGTCACCGGTTAAGCAAGTACAAACAGGAATCGAATGC-3

5’-TGGCTTACTAGTCCATAACTGTCCTTTTAACAGCGATCGC-3’

2. following reagent places on ice, by the low d[X that waits the sudden change degree of preparation in the PCR pipe that fills a prescription] TP error-prone PCR systems 4 pipes:

10×PCR?buffer 5μl

dNTPs 4μl(250μmol/L?each)

Primer?1 1μl(0.5μmol/L)

Primer?2 1μl(0.5μmol/L)

Template 1μl(60ng)

Taq 1μl(5U)

25mmol/L?Mg ²⁺ 0.5μl(0.25mmol/L)

5mmol/LMn ²⁺ 0.5μl(0.05mmol/L)

10mmol/Ld[X]TP 5μl(1000μmol/L)

ddH ₂O To?50μl

3. following reagent places on ice, prepares the d[X of medium sudden change degree by following the prescription in the PCR pipe] TP error-prone PCR systems 4 pipes:

10×PCR?buffer 5μl

dNTPs 4μl(250μmol/L?each)

Primer?1 1μl(0.5μmol/L)

Primer?2 1μl(0.5μmol/L)

Template 1μl(60ng)

Taq 1μl(5U)

25mmol/L?Mg ²⁺ 1μl(0.5mmol/L)

5mmol/L?Mn ²⁺ 1μl(0.1mmol/L)

10mmol/L?d[X]TP 7.5μl(1500μmol/L)

ddH ₂O To?50μl

4. following reagent places on ice, prepares the d[X of high sudden change degree by following the prescription in the PCR pipe] TP error-prone PCR systems 4 pipes:

10×PCR?buffer 5μl

dNTPs 4μl(250μmol/L?each)

Primer?1 1μl(0.5μmol/L)

Primer?2 1μl(0.5μmol/L)

Template 1μl(60ng)

Taq 1μl(5U)

25mmol/L?Mg ²⁺ 2μl(1.0mmol/L)

5mmol/L?Mn ²⁺ 2μl(0.2mmol/L)

10mmol/L?d[X]TP 10μl(2000μmol/L)

ddH ₂O To?50μl

5. above-mentioned 12 pipe prepared PCR reaction system are put into the PCR instrument, press following program setting:

6. fallibility PCR product is mixed by agarose gel electrophoresis (Fig. 1), recovery back and put into 1.5ml EP pipe, the following reagent that thaws on ice utilizes following system to cut 5h at 37 ℃ of enzymes.

7. utilize following system to spend the night in 16 ℃ and connect among the carrier pKH6b that enzyme cuts, coat the kalamycin resistance flat board behind the Transformed E .coli DH5 α, the picking positive colony extracts plasmid.

8. be template with the plasmid that extracts, repeating step 1～7 carries out second and takes turns fallibility PCR.

9. utilize western blot, use the test kit of mouse anti His antibody and invitrogen company, detect the montage activity of the mutein intron that screens.

5. experimental result (Fig. 2)

Through dull and stereotyped preliminary screening to the 7 positive mutant of kalamycin resistance, utilize western blot further to detect as can be known, the protein intron sample 3 that four sudden changes are arranged, 4,6,7 montage activity reach more than 90%, prove successfully to utilize fallibility PCR orthogenesis in various degree to obtain the active protein intron of high montage.

Claims

1. A method utilizing continuous error-prone PCR in various degrees to carry out directed evolution is characterized in that comprising the steps:

(1) Select the target gene as a template to design primers;

(2) Prepare d[X]TP error-prone PCR systems with different degrees of high, medium and low;

(3) Putting the prepared PCR reaction system into a PCR instrument for PCR amplification;

(4) The error-prone PCR product is recovered by agarose gel electrophoresis, digested, connected to the carrier, transformed, screened for positive clones, and the plasmid is extracted,

(5) Using the extracted plasmid as a template, repeat steps (1) to (4) to carry out the second round of error-prone PCR;

(6) Step (5) is repeated, and the corresponding screening system is used to screen useful mutations.

2. a kind of method utilizing different degrees of continuous error-prone PCR to carry out directed evolution according to claim 1, is characterized in that: in the error-prone PCR system of the described low mutation degree of step (2): 25mmol/L The final concentration of Mg ²⁺ is 0.25mmol/L, the final concentration of 5mmol/L Mn ²⁺ is 0.05mmol/L, and the final concentration of 10mmol/L d[X]TP is 1000μmol/L.

3. a kind of method utilizing different degrees of continuous error-prone PCR to carry out directed evolution according to claim 1, is characterized in that: in the error-prone PCR system of the described medium degree of mutation of step (2): 25mmol/L Mg The final concentration of ²⁺ is 0.5mmol/L, the final concentration of 5mmol/L Mn ²⁺ is 0.1mmol/L, and the final concentration of 10mmol/L d[X]TP is 1500μmol/L.

4. a kind of method utilizing different degrees of continuous error-prone PCR to carry out directed evolution according to claim 1, is characterized in that: in the error-prone PCR system of the high degree of mutation described in step (2): 25mmol/L Mg The final concentration of ²⁺ is 1.0mmol/L, the final concentration of 5mmol/L Mn ²⁺ is 0.2mmol/L, and the final concentration of 10mmol/L d[X]TP is 2000μmol/L.

5. A method for directed evolution using different degrees of continuous error-prone PCR according to any one of claims 1-4, characterized in that: the polymerase used is Taq enzyme.

6. A method for directional evolution utilizing continuous error-prone PCR of varying degrees according to claim 1, characterized in that: the PCR reaction conditions described in step (3) are: denaturation and enzyme activation for 3 minutes at 94°C, Denaturation at 94°C for 45 seconds, annealing at 58°C for 30 seconds and 30 seconds, extension at 72°C for 90 seconds, and 35 cycles; finally, extension at 72°C for 10 minutes.

7. a kind of method that utilizes the continuous error-prone PCR of varying degrees to carry out directed evolution according to claim 1, is characterized in that: the primer described in step (1) is template design with the gene of intein Ssp DnaB, Primers are as follows:

Primer 1 5'-AGTCACCGGTTAAGCAAGTACAAACAGGAATCGAATGC-3

Primer 2 5'-TGGCTTACTAGTCCATAACTGTCCTTTTAACAGCGATCGC-3'

8. a kind of method utilizing different degrees of continuous error-prone PCR to carry out directional evolution according to claim 7, is characterized in that: described enzyme cutting site is: Spe I and Age I; Connection carrier is pKH6b; Screening The system is a kanamycin resistant plate.