Molecular cloning, characterization, and expression studies of water buffalo (Bubalus bubalis) somatotropin.
Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species. One interesting variation in the BbST sequence was the replacement of a conserved glutamine residue by arginine. A plasmid was also constructed for the production of BbST in Escherichia coli BL21 (RIPL) CodonPlus, under the control of IPTG-inducible T7-lac promoter. High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies. The inclusion bodies represented over 20% of the E. coli cellular proteins. The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay. The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure-function studies and livestock industry.
Sadaf
S
S
Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan.
Khan
M A
MA
Wilson
D B
DB
Akhtar
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002020:Buffaloes / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors / Q000235:genetics; D013006:Growth Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D051381:Rats; D017386:Sequence Homology, Amino Acid
D013006:Growth Hormone