Functional and biophysical characterization of recombinant human hepatocyte growth factor isoforms produced in Escherichia coli.
Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic cells. Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1) or through kringle 2 (NK2). We report the high-level expression in Escherichia coli of NK1 and NK2, as well as the individual kringle 1 (K1) and N domains of HGF. All proteins accumulated as insoluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gel-filtration steps. Characterization of the purified proteins indicated chemical and physical homogeneity, and analysis by CD suggested native conformations. Although the K1 and N-terminal domains of HGF have limited biological activity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologically active NK1. Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated by mitogenicity, cell scatter, and receptor binding and activation assays. These data indicate that all four bacterially produced HGF derivatives are well suited for detailed structural analysis.
Stahl
S J
SJ
Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bldg 6B, Rm. 1B130, 6 Center Dr., MSC 2775, National Institutes of Health, Bethesda, MD 20892-2775, USA.
Wingfield
P T
PT
Kaufman
J D
JD
Pannell
L K
LK
Cioce
V
V
Sakata
H
H
Taylor
W G
WG
Rubin
J S
JS
Bottaro
D P
DP
D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D017228:Hepatocyte Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor