title: Production of human liver prolidase by Saccharomyces cerevisiae as host cells.
abstract: OBJECTIVE
  To clone and express the recombinant human liver prolidase in yeast and explore the activities of both dipeptidase and organophosphoric acid anhydrolase (OPAA).
  
  
  METHODS
  The cDNA encoding human liver prolidase derived from healthy adult liver was cloned into the pYES2, an expression vector of S cerevisiae, and then transformed into S cerevisiae INVSc1 by electroporation. The transformant with the highest enzymatic activity was induced by galactose for expression. The optimal induction conditions (temperature, induction time, and the initial amount of inoculation cells) were estimated by orthogonal experimental design. The recombinant prolidase and OPAA activities were assayed by spectrocolorimetric methods.
  
  
  RESULTS
  The recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions (20 h, 25 degree, initial OD(600)=0.4), the maximum activities of prolidase and OPAA came to 226.5 and 578 micromol.min(-1).g(-1) protein in cell lysate supernatants, respectively. SDS-PAGE of the recombinant enzyme in disrupted cell supernatants showed a molecular weight of 56 kDa. Intensity scanning of the SDS-PAGE gel revealed that the enzyme accounted for 3.16 % of the total protein in the supernatant. One liter incubation medium produced 7 g of wet yeast cell containing 4.56 mg of the recombination protein.
  
  
  CONCLUSIONS
  The recombinant human liver prolidase produced by yeast cell (S cerevisiae) exhibited both dipeptidase and OPAA activities.
authors.affiliation: Institute of Pharmacology and Toxicology, Academy of Military Medical Science, Beijing 100850, China. sunmj@nic.bmi.ac.cn
authors.forename: Shu-Hao
authors.initials: SH
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Mu-Gen
authors.initials: MG
authors.lastname: Chi
authors.forename: Qing-Ding
authors.initials: QD
authors.lastname: Wang
authors.forename: Man-Ji
authors.initials: MJ
authors.lastname: Sun
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004150:Dipeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D011994:Recombinant Proteins; D004247:DNA; D043303:Aryldialkylphosphatase; D004150:Dipeptidases; C022449:proline dipeptidase