Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies.
The aggregation of recombinant proteins into inclusion bodies is a major problem for expression in bacterial systems. The inclusion bodies must be solubilized and the denatured protein renatured if an active molecule is to be recovered. We have developed such a procedure for the active N-terminal domain of tissue inhibitor of metalloproteinases-2 [TIMP-2-(1-127)], a small mammalian protein containing three disulfide bonds. Conditions for its renaturation were determined by studying the refolding behaviour of reduced and denatured mammalian-cell-expressed TIMP-(1-127) by intrinsic fluorescence. This strategy allows the development of a refolding protocol before generation of a bacterial expression system, and allows rapid and systematic optimization of each refolding variable by assessing its effect on the rate and extent of the refolding reaction. TIMP-(1-127) was expressed at high levels in Escherichia coli, and refolded from TIMP-2-(1-127) inclusion bodies, by means of the method developed with mammalian-cell-expressed protein, to give a refolding efficiency of 30-40% and a final yield of 11-14 mg purified protein/l culture. The chemical structure and conformation of this material was characterized by electrospray mass spectrometry and two-dimensional 1H-NMR; no significant differences were found between it and the native protein. Mass analysis of uniformly 13C-labeled and 15N-labeled protein was used to help identify a mistranslated TIMP-(1-127) contaminant in the purified refolded sample. This technique provides additional information on the nature of the modification and allows a distinction to be made between those modifications that are cell derived, and those that arise from subsequent handling of the protein.
Department of Biosciences, University of Kent, Canterbury, UK.
R A
RA
Williamson
D
D
Natalia
C K
CK
Gee
G
G
Murphy
M D
MD
Carr
R B
RB
Freedman
D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000737:chemistry*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2
D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2