PROTEIN EXPRESSION AND PURIFICATION 11, 169–178 (1997) ARTICLE NO. PT970784 Refolding of a Recombinant Collagen-Targeted TGF-b2 Fusion Protein Expressed in Escherichia coli 1 Bo Han,* Frederick L. Hall,† and Marcel E. Nimni* ,2 *Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, and Divisions of Surgical and †Cardiothoracic Research, Children’s Hospital Los Angeles, Los Angeles, California 90027 Received February 3, 1997, and in revised form June 9, 1997 peptides, which function as potent regulators of cell In this study, a tripartite transforming growth fac- growth, differentiation, and extracellular matrix depo- tor-b (TGF-b2) fusion protein bearing an N-terminal sition (1,2). There are three major TGF-b isoforms in purification tag and an auxiliary collagen binding mammalian cells, designated TGF-b1, TGF-b2, and decapeptide has been constructed and expressed at TGF-b3 (3). The sequence homology among these iso- high levels in Escherichia coli. The resulting recom- forms is greater than 65% and their biological activity binant protein accumulates in an insoluble and bio- is similar in many in vitro assays (4). However, there logically inactive inclusion–body complex. The in- are significant differences in potency and physiological soluble protein was solubilized in guanidine hydro- effects, including inhibition of hematopoeitic stem cells, chloride and a Ni-chelating affinity column was regulation of endothelial cells proliferation, and induc- utilized to isolate the 13.5-kDa TGF-b2 fusion pro- tion of mesoderm formation in vertebrate embryos (4 – tein, which was then refolded into its native confor- 6), suggesting that particular TGF-b isoforms may be mation under controlled redox conditions. The for- potentially useful for specific therapeutic applications. mation of native homodimers was monitored by non- Currently, two approaches are used to obtain TGF- reducing sodium dodecyl sulfate–polyacrylamide b1 for biochemical and pharmacological studies. One gel electrophoresis gradient gels and the bioactivity involves extraction from tissues, such as placenta, determined by a quantitative TGF-b assay system us- blood platelets, or bone. However, such biological ing mink lung epithelial cells transfected with a plas- sources are limited, especially for the minor isoforms minogen activator inhibitor-1 promoter/luciferase like TGF-b2 and b3 (3). Another approach involves the reporter plasmid. To optimize yields, renaturation conditions including denaturants, limiting protein expression and purification of recombinant TGF-b1 concentrations, redox ratios, dialysis conditions, from mammalian CHO cells (7). Although expression and refolding kinetics were studied and monitored of recombinant proteins in transformed prokaryotic mi- by bioactivity. These studies demonstrate that re- croorganisms could, theoretically, serve to guarantee combinant TGF-b2 fusion proteins can be produced an unlimited supply of such recombinant proteins, un- in E. coli and renatured into biologically active ho- fortunately they have not generated native, soluble, modimers. Furthermore, they confirm that the auxil- and biologically active conformations. Instead, overex- iary collagen binding domain effectively targets the pression of recombinant protein from Escherichia coli recombinant growth factor to type I collagen. Taken often results in the accumulation of insoluble proteins together, these studies advance the technology nec- within bacterial inclusion bodies (8) due in part to the essary to generate large quantities of targeted TGF- b fusion proteins for specific biomedical applica- tions.  1997 Academic Press D-thiogalactopyranoside; PMSF, phenylmethysulfonyl fluoride; VWF, von Willebrand factor; TFA, trifluoroacetic acid; PAI-1, plas- TGF-b 3 is a multifunctional growth factor, part of a minogen activator inhibitor-1; HAc, acetic acid; PEG, polyethylene large and growing family of structurally related poly- glycol; L-Arg, L-arginine. CHO, Chinese hampster ovary; RT-PCR, reverse transcriptase-polymerase chain reaction; BSA, bovine serum albumin; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Ea- 1 This work was supported by NIH AG Grant 02577. 2 To whom correspondence should be addressed. gle’s medium; PBS, phosphate-buffered saline; DTT, dithiothreitol; SDS – PAGE, sodium dodecyl sulfate – polyacrylamide gel electropho- 3 Abbreviations used: TGF-b, transforming growth factor-b; GSH, reduced glutathione; GSSG, oxidized glutathione; IPTG, isopropyl b- resis; DMF, dimethylformamide; LAP, latency-associated peptide. 169 1046-5928/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.