Expression vector of nuclease protein Cas9 and construction method and expression purification of expression vector of nuclease protein Cas9

Abstract

The invention discloses an expression vector of a nuclease protein Cas9 and a construction method and expression purification of the expression vector of the nuclease protein Cas9. According to the expression vector of the nuclease protein Cas9, an original thioredoxin sequence in pET32a is replaced with cDNA of an amplified thioredoxin-histidine-tag-tobacco-etch-virus-protease recognition site, and codons are optimized to a Cas9 gene sequence which is easy to express in a host and has nuclear localization signal peptide and 3xFLAG tag sequence fused in a C-terminal to be integrated into the thioredox in sequence. According to the expression vector of the nuclease protein Cas9 and the construction method and expression purification of the expression vector of the nuclease protein Cas9, codon optimization and thioredoxin fusion expression are used for obtaining a large amount of the nuclease Cas9 protein, under the condition that only an LB medium is used, 10 mg of a highly purified active Cas9 protein can be finally obtained per gram of escherichia coli Tuner (DE3) wet bacteria, and high expression of the high-fidelity nuclease protein Cas9 at a low cost is realized; and the requirements on the host are not high, and the obtained protein can be used for large-scale biological in vitro assembly and in vivo gene editing experiments.

Description

technical field [0001] The invention belongs to the technical field of expression vector construction, and relates to the construction of a nuclease protein expression vector, in particular to an expression vector of the nuclease protein Cas9, a construction method and expression purification thereof. Background technique [0002] CRISPR (Clustered regularly interspaced short palindromic repeats) is called regularly clustered interspaced short palindromic repeats, derived from bacteria, is a system for bacteria to protect themselves against viruses, and can be widely used to delete, add, activate or inhibit various organisms target gene. Thus, CRISPR-Cas9 is a precise and versatile genetic weapon. [0003] According to "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wideoff-target effects", four amino acid mutations of Streptococcus pyogenes Cas9 (SpCas9) can reduce the off-target effect of its gene editing, but the mutation sites are N497 The protease activ...

AI Technical Summary

Problems solved by technology

However, due to the presence of rare codons in the transcription and translation of nucleic acid sequences, this expression requires a specific host bacterium; and a large amount of culture medium is required to obtain the protein Cas9, which is costly; and three systematic purification steps are required to obtain a relatively pure Target protein, complex operation (Anders C,Jinek M.Invitro enzymology of Cas9[J].Methods Enzymol,2014,546:1-20.)

In addition, the market price of Cas9 protein is extremely high, so the low expression of Cas9 protein limits the popularity of Cas9 system assembly in vitro and editing in vivo

Images