Method of modifying gene and obtained recombinant gene and encoded protin

Abstract

This invention relates to a method for gene directed evolution, specifically, a method for gene recombination and application. The method comprises the steps of: dividing the reaction into two groups, performing recombination, mixing the two reactants, and performing a series of PCR reactions to realize gene recombination. The sequencing results show that the method is better than previous gene recombination methods, and is especially suitable for recombination of genes with low sequence identity. The effectivity of the method is testified by using adenosylmethionine synthetase genes from Escherichia coli and yeast as the starting genes, and can obtain high activity recombined enzyme.

Description

technical field [0001] The invention relates to a gene-guided evolution method, specifically a gene shuffling method and its application, and is a method especially suitable for inter-gene shuffling with low homology. Background technique [0002] Guided evolution is artificially creating special evolutionary conditions in the laboratory, simulating natural evolutionary mechanisms (such as mutations, recombination), and directional selection of 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) are especially applied to improve enzyme activity (Crameri, A., Raillard, S-A., Bermudez, E.&Stemmer, W.P.C. DNAshuffling of a family of genes from diverse...

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Problems solved by technology

However, when sequences with less than 70% homology are shuffled by this method, the products tend to be heavily inclined to form repolymerized strands of the parent strands

Although thereafter some recombination methods relying on sequence homology (Ostermeier, M., Shim, J.H. & Benkovic, S.J.A combinatorial approach to hybridenzymes independent of DNAhomology. Nat. Biotechnol. 1999, 17, 1205-1209.; Zhao, H., Giver, L., Shao, Z. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 1998, 16, 258-261.; Jon, E. et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat. Biotechnol. 2002, 20, 1251-1255.) was proposed, but none of them can fundamentally solve this problem

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