title: Expression, refolding and purification of Ov-GRN-1, a granulin-like growth factor from the carcinogenic liver fluke, that causes proliferation of mammalian host cells.
abstract: Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver fluke of humans, Opisthorchis viverrini, and showed that recombinant Ov-GRN-1 expressed and refolded from Escherichia coli caused proliferation of mammalian cell lines at nanomolar concentrations. We now report on an optimized method to express and purify monomeric Ov-GRN-1 in E. coli using a straightforward and scalable purification and refolding process. Purified monomeric protein caused proliferation at nanomolar concentrations of cancerous and non-cancerous cell lines derived from human bile duct tissue. The expression and purification method we describe herein will serve as a backbone upon which to develop expression and purification processes for recombinant GRNs from other organisms, accelerating research on this intriguing family of proteins.
authors.affiliation: Queensland Tropical Health Alliance, James Cook University, Cairns QLD 4878, Australia.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Smout
authors.forename: Jason P
authors.initials: JP
authors.lastname: Mulvenna
authors.forename: Malcolm K
authors.initials: MK
authors.lastname: Jones
authors.forename: Alex
authors.initials: A
authors.lastname: Loukas
mesh_terms: D000818:Animals; D001652:Bile Ducts / Q000166:cytology / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000209:etiology* / Q000378:metabolism / Q000469:parasitology / Q000473:pathology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D008113:Liver Neoplasms / Q000209:etiology* / Q000378:metabolism / Q000469:parasitology / Q000473:pathology; D009889:Opisthorchiasis / Q000150:complications* / Q000378:metabolism / Q000469:parasitology / Q000473:pathology; D009891:Opisthorchis / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids; D000077153:Progranulins; D055503:Protein Multimerization; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D014169:Transformation, Bacterial
chemical_list: D015801:Helminth Proteins; D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins; D011993:Recombinant Fusion Proteins