title: Optimal conditions for expressing a complement component 3b functional fragment (α2-macroglobulin receptor) gene from Epinephelus coioides in Pichia pastoris.
abstract: The α2-macroglobulin receptor (α2MR) is a major domain of complement component 3b, which may play an important role in regulating the downstream complement system in teleosts. In order to characterize the domain thoroughly larger than currently available quantities are required. Thus, in this study the Epinephelus coioides α2MR (Ec-α2MR) was expressed and secreted by the methylotrophic yeast Pichia pastoris with variations in pH and induction time to identify optimal production conditions. At pH 5.5 with 48h induction 13mg of Ec-α2MR (ca. 90% purity) was obtained from 500ml of culture. The Ec-α2MR protein product was validated by MALDI-TOF MS sequence analysis, and both Western blotting and ELISAs demonstrated that it possessed the expected activity, binding to C3b or C3b homolog antibodies, and thus can be used for future studies of the interactions and functions of complement proteins in teleosts.
authors: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, PR China.
authors: Gui-Hong
authors: GH
authors: Cha
authors: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, PR China.
authors: Sheng-Wei
authors: SW
authors: Luo
authors: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, PR China.
authors: Zeng-Hua
authors: ZH
authors: Qi
authors: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, PR China.
authors: Yuan
authors: Y
authors: Liu
authors: Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, PR China. Electronic address: wangwn@scnu.edu.cn.
authors: Wei-Na
authors: WN
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001492:Bass / Q000235:genetics*; D015153:Blotting, Western; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D003179:Complement C3b / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
chemical_list: D000906:Antibodies; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011994:Recombinant Proteins; D003179:Complement C3b
keywords: Epinephelus coioides; Pichia pastoris; Protein expression; α2-Macroglobulin receptor