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.
Copyright © 2011 Elsevier Inc. All rights reserved.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Bile Ducts / cytology
-
Bile Ducts / drug effects
-
Carcinoma, Hepatocellular / etiology*
-
Carcinoma, Hepatocellular / metabolism
-
Carcinoma, Hepatocellular / parasitology
-
Carcinoma, Hepatocellular / pathology
-
Cell Line
-
Cell Proliferation / drug effects
-
Chromatography, Affinity
-
Cloning, Molecular
-
Electrophoresis, Polyacrylamide Gel
-
Escherichia coli
-
Helminth Proteins / chemistry
-
Helminth Proteins / genetics
-
Helminth Proteins / isolation & purification*
-
Helminth Proteins / metabolism
-
Helminth Proteins / pharmacology
-
Humans
-
Inclusion Bodies / chemistry
-
Inclusion Bodies / metabolism
-
Intercellular Signaling Peptides and Proteins / chemistry
-
Intercellular Signaling Peptides and Proteins / genetics
-
Intercellular Signaling Peptides and Proteins / isolation & purification*
-
Intercellular Signaling Peptides and Proteins / metabolism
-
Intercellular Signaling Peptides and Proteins / pharmacology
-
Liver Neoplasms / etiology*
-
Liver Neoplasms / metabolism
-
Liver Neoplasms / parasitology
-
Liver Neoplasms / pathology
-
Opisthorchiasis / complications*
-
Opisthorchiasis / metabolism
-
Opisthorchiasis / parasitology
-
Opisthorchiasis / pathology
-
Opisthorchis / growth & development
-
Opisthorchis / metabolism*
-
Plasmids
-
Progranulins
-
Protein Multimerization
-
Protein Refolding
-
Recombinant Fusion Proteins / chemistry
-
Recombinant Fusion Proteins / genetics
-
Recombinant Fusion Proteins / isolation & purification*
-
Recombinant Fusion Proteins / metabolism
-
Recombinant Fusion Proteins / pharmacology
-
Transformation, Bacterial
Substances
-
Helminth Proteins
-
Intercellular Signaling Peptides and Proteins
-
Progranulins
-
Recombinant Fusion Proteins