Integrated gene recombined process, recombined gene and coding protein obtained thereby

Abstract

The invention relates to an integrated gene repacking method. First to select the preparing mutation or repacking linear dual-chain gene and amplify 15 circulates by the PCR and then the product is deposited by the 80% alcohol and remove the soluble component; next the DNA polymerase, the dNTP, the buffer and the ultrapure water are added into the deposition to compose the StEP system and amplifyto get the repacking gene. The objective DNA fragments are amplified especially by the sepharose gel electrophoresis, then to express and select to get the repacking gene. The invention can reach thegene mutation and repacking in a process and detected by the synzyme gene sam1, so it gets the recombined enzyme to improve the accumulation of the SAM.

Description

technical field [0001] The present invention relates to the guided evolution of genes, specifically a comprehensive gene shuffling method and the obtained recombinant gene and encoded protein, and is a kind of gene shuffling that can skillfully integrate error-prone PCR and StEP in one reaction process method. Background technique [0002] Guided evolution is to artificially create special evolutionary conditions in the laboratory to simulate natural evolutionary mechanisms (such as mutation and recombination), so as to select protein molecules with desired properties without knowing the spatial structure and function of proteins. At present, this mechanism has been widely used in protein medicine, agriculture, chemical industry, bioengineering and other fields (Patten, P.A., Howard, R.J. & Stemmer, W.P.C. Applications of DNA shuffling to pharmaceuticals and vaccines.Curr Opin Biotechnol.1997,8:724 -733), especially used to improve enzyme activity (Crameri, A., Raillard, S-...

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Problems solved by technology

[0004] In previous studies, StEP and error-prone PCR were often used in combination, but they needed to go through two relatively independent reaction processes (Zhao, H., Giver, L., Shao, Z. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitrorecombination.Nat.Biotechnol.1998,16,258-261.; Zhao H, Arnold F H.Directed evolution converts subtilisin E into a functional equivalent ofthermitase[J].Protein Eng,1999,12:47 ~53.), and these processes are often very labor-intensive and time-consuming, and in these cumbersome steps, some meaningful mutation sites may be lost

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