title: cDNA isolation and functional characterization of squalene synthase gene from Ornithogalum caudatum.
abstract: As the first step of ongoing efforts to investigate the genes responsible for the biosynthesis of steroidal saponins in the medicinal plant Ornithogalum caudatum, this investigation reported the cDNA isolation, prokaryotic expression and functional characterization of squalene synthase (SQS) gene from O. caudatum for the first time. Specifically, two unigenes showing high sequence identity to SQS were retrieved from RNA-Taq data, and then a full-length OcSQS1 corresponding to the two unigenes was isolated from O. caudatum genome by a nested PCR assay. The open reading frame of OcSQS1 was 1230 bp and encoded a polypeptide of 409 aa. OcSQS1 was predicted to be a membrane-bound protein with at least four conserved motifs associated with binding, regulatory and catalytic activities of OcSQS1 and two transmembrane domains. Next, many attempts to generate soluble OcSQS1 in heterologous Escherichia coli were made, including optimization of expression conditions, application of varied expression plasmids with different tags, secretory peptides and molecular chaperones, and truncated mutation of OcSQS1. Finally, the successful availability of a soluble, truncated OcSQS1 mutant was achieved by combinational use of the utensils from the vast genetic toolbook. Moreover, this truncated OcSQS1 mutant retained the folding capability as well as its catalytic activity, converting FPP to form squalene. Importantly, the present research tentatively verified the involvement of the second transmembrane domain in the proper folding of the recombinant OcSQS1 protein.
authors.affiliation: Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.affiliation: Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China.
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Li
authors.affiliation: Beijing Institute of Metrology, Beijing, 100029, China.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Pan
authors.affiliation: Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China. Electronic address: jianqiangk@imm.ac.cn.
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Kong
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D031442:Ornithogalum / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D013186:Farnesyl-Diphosphate Farnesyltransferase
keywords: Escherichia coli; Ornithogalum caudatum; Soluble expression; Squalene synthase