title: Effects of co-expression of molecular chaperones on heterologous soluble expression of the cold-active lipase Lip-948.
abstract: The cold-active lipase gene Lip-948, cloned from Antarctic psychrotrophic bacterium Psychrobacter sp. G, was ligated into plasmid pColdI. The recombinant plasmid pColdI+Lip-948 was then transformed into Escherichia coli BL21. SDS-PAGE analysis showed that there was substantive expression of lipase LIP-948 in E. coli with a yield of about 39% of total protein, most of which was present in the inclusion body. The soluble protein LIP-948 only consisted of 1.7% of total LIP-948 with a specific activity of 66.51U/mg. Co-expression of molecular chaperones with the pColdI+Lip-948 were also carried out. The results showed that co-expression of different chaperones led to an increase or decrease in the formation of soluble LIP-948 in varying degrees. Co-expression of pColdI+Lip-948 with chaperone pTf16 and pGro7 decreased the amount of soluble LIP-948, while the soluble expression was enhanced when pColdI+Lip-948 was co-expressed with "chaperone team" plasmids (pKJE7, pG-Tf2, pG-KJE8), respectively. LIP-948 was most efficiently expressed in soluble form when it was co-expressed with pG-KJE8, which was up to 19.8% of intracellular soluble proteins and with a specific activity of 108.77U/mg. The soluble LIP-948 was purified with amylase affinity chromatography and its enzymatic characters were studied. The optimal temperature and pH of LIP-948 was 35°C and 8, respectively. The activity of LIP-948 dropped dramatically after incubation at 50°C for 15min and was enhanced by Sr(2+), Ca(2+). It preferentially hydrolyzed 4-nitrophenyl esters with the shorter carbon chain.
authors.affiliation: First Institute of Oceanography, SOA, Qingdao 266061, China.
authors.forename: Cui
authors.initials: C
authors.lastname: Shuo-shuo
authors.forename: Lin
authors.initials: L
authors.lastname: Xue-zheng
authors.forename: Shen
authors.initials: S
authors.lastname: Ji-hong
mesh_terms: D000864:Antarctic Regions; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005786:Gene Expression Regulation; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009596:Nitrophenols / Q000032:analysis / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D044096:Psychrobacter / Q000737:chemistry / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; C471213:His-His-His-His-His-His; D018832:Molecular Chaperones; D009596:Nitrophenols; D009842:Oligopeptides; D011994:Recombinant Proteins; C024719:4-nitrophenyl; D006639:Histidine; D008049:Lipase