Construction Of S-adenosylhomocysteine Hydrolase Gene Engineering Bacteria And Activity Analysis

S-adenosylhomocysteinehydrolase(SAHH) plays an important role in the regulation of methyl circulating metabolism in eukaryotic cells, it's a proteolytic enzyme that regulates the methylation of methyl groups in the presence of biological cells, this proteolytic enzyme in humans, mammals, plants, fungi has been widely studied. Its main function is to catalyze the reversible hydrolysis of S-adenosylhomocysteine(SAH) to generate adenosine(Ado) and homocysteine(Hcy).In vitro, this balance tends to generate SAH. The research has indicated that the level of SAHH in the human body can affect the incidence of coronary heart disease and Alzheimer's disease.In order to study the proteolytic enzymes better, we use yeast Pichia pastoris expression of the target protein SAHH, because the yeast Pichia pastoris is currently being studied in eukaryotic cells are mature model microorganisms, so we will integrate this hydrolase protein gene into Pichia pastoris genome by cloning technology.The specific experimental programs are as follows: We research human source encode the S-adenosylhomocysteinehydrolase gene from GenBank, designed the enzyme cutting site and recombinant His-tag before and after the gene, And then synthesis the complete sahh gene coding frame, through the whole gene synthesis technology, construct the expression vector pPIC9K-sahh, as the gene was digested by tail enzyme Bgl II, it was integrated into the genome of Pichia pastoris GS115 by electroporation, screened high copy recombinant by geneticin G418, recombinant phenotypes were identified as Mut S and the bacterial PCR was identified as positive,then the positive was induced by 1% methanol to secrete SAHH protein. And the supernatant use chromatography Ni-NTA to purify. This method has the advantages of convenient screening, stable expression of protein products and easy purification.Human sahh gene contains 1299 base pairs, it encodes 432 amino acids, and the relative molecular weight is about 47.5 kDa, and the expression product was about 48 kDa by SDS-PAGE. As SAH was further hydrolyzed by SAHH to homocysteine andadenosine, and homocysteine can be directly quantified by Ellman reagent, the absorbance increased with the increase of the TNB under the 412 nm. We measured SAHH protease activity was 626.6 IU/mL. And after Ni-NTA affinity chromatography purification SAHH activity was 1106.8 IU/mL, sahh gene was not integrated into the genome of Pichia pastoris GS115 product was obtained through the method of fermentation protease activity was 1.33 IU/mL, so we can ignore.In conclusion: SAHH was highly secreted expression in Pichia pastoris. we can compare with the difference structure in S-adenosylhomocystein nucleosidase(SAHN)in the pathogen, screen the natural inhibitors which inhibit the SAHN enzyme activity but not affect the SAHH activity.