High-level expression of ovine growth hormone in Escherichia coli: single-step purification and characterization.
The gene for ovine growth hormone (oGH) was expressed without signal sequences in Escherichia coli. A recombinant plasmid expression vector has been constructed which directs the synthesis of a fusion protein containing a stretch of six histidine residues (His6) at the amino-terminus under the control of a T5 promoter. Upon induction with isopropyl-beta-D-thiogalactopyranoside, the recombinant protein was synthesized and accumulated in the cytoplasm in the form of inclusion bodies, at levels of approximately 18% of the total cellular protein. The recombinant ovine growth hormone containing His tag was recovered and purified to >95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity Ni2+.NTA resin that has selectivity for proteins with neighboring histidine residues. Characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and amino terminal analysis demonstrated the authenticity of the fusion protein. The purified RoGH after refolding was found to be functionally active in terms of its receptor binding and antigenicity as analyzed by radio receptor assay and radio immuno assay. Yields of the purified expressed protein were found to be 32 microg/ml at a shake-flask level. Thus, results indicate that a combination of E. coli expression and affinity purification by Ni2+.NTA chromatography promises to be a rapid method to produce oGH for use in structure-function studies.
Appa Rao
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KB
Embryo Biotechnology Laboratory, Gene Regulation Laboratory, Product Development Cell, National Institute of Immunology, JNU Complex, Aruna Asaf Ali Marg, New Delhi-110, 067, India.
Garg
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LC
Panda
A K
AK
Totey
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SM
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D013006:Growth Hormone / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine; D002479:Inclusion Bodies; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D011863:Radioimmunoassay; D011986:Receptors, Somatotropin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D017421:Sequence Analysis; D012756:Sheep
D010455:Peptides; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D013006:Growth Hormone