[Expression of glutathione S-transferase zeta class genes in Saccharomyces cerevisiae].
The zeta class of glutathione S-transferase (GSTZs), which is an important multifunctional enzyme, relates to the cell metabolism and contamination elimination. The GSTZ genes from Arabidopsis thaliana L. and Brassica napus L. cv. Shan 2B and Ken C1 were cloned into the multi-cloning site of the shuttle expression vector pYES2. After the recombinants were obtained, the recombinant plasmids were isolated and introduced into the defective mutant INCSc1 of Saccharomyces cerevisiae. Then the recombinant strains Y2At, Y2BnB and Y2BnC were obtained after cultured on SC-U selective plates. When induced in the medium containing galactose and maltose, the recombinant yeast expressed as active GSTZs showing the dichloroacetic acid dechlorinating activity, which existed in the yeast cell as a soluble state. The comparison of different carbon sources showed that sucrose and glucose significantly exhibited the expression of GSTZ gene; glycerol somewhat affected the growth of yeast but increased the specific activity of GSTZ by 17%; and galactose slightly affected the yeast growth with no affection to the activity of GSTZ. Zero to ninety-six hrs induction experiments showed that specific activity of GSTZ in recombinant yeast reached highest when induced for 36 hours. The specific activity of AtGSTZ, BnGSTZ-B and BnGSTZ-C was 5.3 U/mg, 4.3 U/mg and 0.3 U/mg, respectively. The values are lower than that expressed in the E. coli and wheat-sperm cell-free protein synthesis system. However, the relative activity of three sources was similar in E. coli and wheat cell free system. The Km value of GSTZ genes from different sources was 0.59 mmol/L and 0.79 mmol/L for AtGSTZ and BnGSTZ-B, respectively, suggesting the GSTZ enzyme from Abrabidopsis thaliana has higher affinity to DCA than that from Brassica napus.
Jia
Xiang-Dong
XD
Key Laboratory of Bioactive materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China. xiangdong_jia@sohu.com
Chen
Xi-Wen
XW
Chen
De-Fu
DF
Chen
Jie
J
D017360:Arabidopsis / Q000201:enzymology*; D029688:Brassica napus / Q000201:enzymology*; D003001:Cloning, Molecular; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D010940:Plant Proteins; D005982:Glutathione Transferase