中国生物工程杂志

In order to study its specificities,the NotⅠR gene was cloned from Nocardia otitidis-caviarum. Firstly,the genome DNA of Enterobacter agglomerans was extracted as template,obtained EagⅠmethylase gene by PCR and connected EagⅠM gene to pBR322 vector to gain recombinant expression plasmid pBR322-Eag ⅠM. Then transformed this plasmid into E. coli 2566. Secondly,extracted the genome DNA from Nocardia otitidis-caviarum as template and obtained the restriction enzyme NotⅠR gene by PCR. After ligating the NotⅠR gene to pACYC184-PT₇,the pACYC184-PT₇-NotⅠR plasmid was transformed into the ER2566 which was protected through the methylation by pBR322-EagⅠM recombinant plasmid. The engineered strain ER2566 could be induced to express restriction enzymes NotⅠ by IPTG and the induction conditions were optimized to make its expression mostly in soluble form. The enzyme was purified by ÄKTA purifier 100 protein purification system. Through DEAE Sephrose FF,phenyl HP and Superdex 75 10/300 GL molecular sieve chromatography,the Not Ⅰenzyme was purified 35-fold,the yield was 9.8 × 10⁶ Units / g wet cell which was up to17.8% of the crude enzyme and the specific activity of the purified NotⅠwas 1.37 × 10⁶U/mg. Digestion results showed that the enzyme was purified to homogeneity and was free of detectable contamination by other DNase(exo and endo). After optimization of the expression and purification conditions,the yield and efficiency of NotⅠ enzyme were greatly improved in comparison with that previously reported.