title: cDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli.
abstract: Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation.
authors.affiliation: College of Biological Engineering, Jimei University, Xiamen 361021, PR China.
authors.forename: Long
authors.initials: L
authors.lastname: Han
authors.affiliation: College of Biological Engineering, Jimei University, Xiamen 361021, PR China.
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
authors.affiliation: College of Biological Engineering, Jimei University, Xiamen 361021, PR China.
authors.forename: Chao-lan
authors.initials: CL
authors.lastname: Shi
authors.affiliation: College of Biological Engineering, Jimei University, Xiamen 361021, PR China.
authors.forename: Xiao-Nan
authors.initials: XN
authors.lastname: Wei
authors.affiliation: College of Biological Engineering, Jimei University, Xiamen 361021, PR China.
authors.forename: Huan
authors.initials: H
authors.lastname: Li
authors.affiliation: College of Biological Engineering, Jimei University, Xiamen 361021, PR China. Electronic address: cuihongdu@jmu.edu.cn.
authors.forename: Cui-Hong
authors.initials: CH
authors.lastname: Du
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002347:Carps / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009210:Myofibrils / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D005956:Glucose-6-Phosphate Isomerase
keywords: Crucian carp; Escherichia coli; Glucose-6-phosphate isomerase; Myofibril-bound serine proteinase inhibitor; Recombinant expression