Purification and characterization of recombinant truncated human interleukin-11 expressed as fusion protein in Escherichia coli.
Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.
Tan
Haidong
H
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, 116023 Dalian, PR China. hdt@dicp.ac.cn
Dan
Guoping
G
Gong
Huiying
H
Cao
Lijun
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D017370:Interleukin-11 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein
D017370:Interleukin-11; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase