Purification And Its Clinical Application Of Humanized S-Adenosyl-L-Homocysteine Hydrolase

Objective:based on the successful construction of Escherichia BL21(DE3) harbouring expression vector PQE30, through the mass culture of the bacteria to express SAHH, which was purified by chromatographic methods, our experiments were in order to obtain high purity of active SAHH, establish a complete purification scheme and preliminarily investigated its application in biological and chemical reagents.Methods:1. E.coli BL21(DE3) were grown in 1-liter LB medium containing 50ug/ml ampicillin at 37℃and at the rotation speed of 200rpm. Isopropyl-β-D-galactopyranoside (0.5mM) was added to the culture when the OD600 reached about 0.5, and the bacteria were grown for 10h.2. The bacteria were lysised by homogenization, and the crude products of SAHH were obtained by ammonium sulfate fractionation. Then Sepharose CL-6B Fast Flow,DEAE Sepharose Fast Flow and Sephadex G-75 column chromatography were used finally.3. The products of expression and purification were identified by SDS-PAGE electrophoresis and Western blotting.4. According to the enzymatic determination of circulating Hcy, under the same conditions, Hcy levels in the same serum were respectively detected by the known reagent and the substituted reagent, and the differences were compared by paired t test.Results:1. E.coli BL21(DE3) induced by 0.5mmol/L IPTG successfully expressed SAHH at 37℃and at the shaking speed of 200rpm when the bacterium were grown 10h.2. SAHH was purified by chromatography. By SDS-PAGE electrophoresis, the purified product was found near a single protein. The purified enzyme preparation for implementation of PVDF transfer membrane after electrophoresis, a single band was found in the transfer membrane.3. The difference between the detection results of the known reagent and that of the substituted reagent was not statistically significant, p>0.05.Conclusions:1. SAHH was successfully expressed by a classic E.coli expression system, and there was stable expression, easy operation and low pollution.2. SAHH was successfully purified, the SAHH kept stable and there was no degradation in the process of the purification.3. Our study confirmed that the purified SAHH has some activity and has certain application value in biochemical reagent.