Cloning Restriction Endonuclease Dpn Ⅰ And Expression In Pichia Pastorios And Its Quality Research

Restriction enzyme Dpn Ⅰ, also known as DNA adenine methylase.It is a kind of enzyme which can split in a limited number of specific parts of the DNA molecule.This enzyme’s name is:EC3.1.21.4.The Enzyme gene’s total length is754bp, encoding254amino acids.Restriction endonuclease enzyme Dpn Ⅰ comes from Diplococcus pneumoniae.Recognition sequence and cleavage site is5’...GAm/TC...3’.It is mainly used for biochemical studies, genetic engineering, genetic engineering tools enzyme.It is most commonly used in PCR-based gene-directed mutagenesis experiments.In this experiment, we firstly acquired Dpn Igenes, and then clone it into the Pichia yeast secretion-type expression vector pHBM905A, and the inside cutting enzymes of SalI linearized the recombinant plasmid,and then it was switch into the Pichia pastoris yeast strain GS115by electroporation. After MD plates screening and PCR,correct transformants was identified.Then we did the shaking flask culture to recombinant GS115strain which was carrying Dpn I genes, induced by methanol, remove supernatant,and used it do SDS-PAGE analysis,the result showed that the protein was expressed, and acquired a large number of high-expression strain by screening.High expression levels in Shaking flask culture appeared in in the fifth, but the nature of the enzyme molecular weight has changed.Molecular weight is about66KD, bigger than the predicted protein molecular weight, the optimum reaction temperature is60℃, enzyme activity still remained60%in95℃, the thermal stability is improved, the optimum pH value is7.6. These occurred changes may be due to the impact of glycosylation.Glycosylation kit detected the protein is highly glycosylated. After the protein was deglycosylated,the protein molecular weight reduced to29KD, this size is consistent with the theory predicted molecular weight of this protein. Dpn Ⅰ successful expressed in Pichia pastoris.After the protein was purified by heparin,enzyme digestion experiments show that the Dpn Ican specific cut plasmid which was methylated by colibacillus. it proved that recombinant Dpn Ⅰ protein has biologically activity.The same time, we made Dpn I’s open reading frame (ORF) and the open reading frame (ORF) from the sulfur mine solfataricus Sso7d genes do N-terminal fusion, and using the Pichia expression system to fusion protein expression.Fusion recombinant protein was successfully expressed.In further work, we are prepared to use the high heat-resistant activity Dpn I enzyme expressed, in process of PCR-based gene-directed mutagenesis experiments, use this enzyme in the PCR step, to achieve elimination of the template in the PCR amplification process,it makes the amplification products can be directly converted,and get the purpose of mutagenesis product.Not need to go through the traditional steps:firstly recycling,Dpn Ienzyme digestion and then transformation.It makes the Gene mutagenesis steps to become better simplified, convenient and provincial.