title: Secretory expression and characterization of the recombinant myofibril-bound serine proteinase of crucian carp (Carassius auratus) in Pichia pastoris.
abstract: The myofibril-bound serine proteinase (MBSP) is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin and was thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo. In order to better understand the characteristic differences between native MBSP and recombinant MBSP (rMBSP) and to obtain large quantity of MBSP for its application in protein science study, the crucian carp MBSP gene was cloned (669 bp) and expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks, and 66.85 mg rMBSP/L in the fermentation supernatant was obtained. SDS-polyacrylamide gel electrophoresis (PAGE) showed a main protein band with molecular weight of approximately 36 kDa. Substrate specificity analysis revealed that the rMBSP specifically cleaved substrates at the carboxyl side of lysine residue which differed from native MBSP that cleaved substrates at the carboxyl side of arginine and lysine residues. The optimum temperature and optimum pH range of the rMBSP were 55 °C and pH7.5, respectively. Furthermore, similar to native MBSP, the rMBSP also revealed high thermostability and pH stability and is effective in degradation of myofibrillar proteins from the skeletal muscle of crucian carp.
authors: College of Biological Engineering, Jimei University, Xiamen 361021, PR China.
authors: Cui-Hong
authors: CH
authors: Du
authors: Long
authors: L
authors: Han
authors: Qiu-Feng
authors: QF
authors: Cai
authors: Min-Jie
authors: MJ
authors: Cao
mesh_terms: D000818:Animals; D002347:Carps / Q000378:metabolism*; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004795:Enzyme Stability; D029941:Fish Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009210:Myofibrils / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014170:Transformation, Genetic
chemical_list: D029941:Fish Proteins; D011994:Recombinant Proteins; D057057:Serine Proteases