Renaturation of SPARC expressed in Escherichia coli requires isomerization of disulfide bonds for recovery of biological activity.
SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin and BM-40) belongs to a group of secreted macromolecules that modulate cellular interactions with the extracellular matrix. During vertebrate embryogenesis, as well as in tissues undergoing remodeling and repair, the expression pattern of SPARC is consistent with a fundamental role for this protein in tissue morphogenesis and cellular differentiation. Human SPARC was cloned by the polymerase chain reaction from an endothelial cell cDNA library and was expressed in Escherichia coli as a biologically active protein. Two forms of recombinant SPARC (rSPARC) were recovered from BL21(DE3) cells after transformation with the plasmid pSPARCwt: a soluble, monomeric form that is biologically active (Bassuk et al., 1996, Archiv. Biochem. Biophys. 325, 8-19), and an insoluble form sequestered in inclusion bodies. Aggregated rSPARC was unfolded by urea treatment, purified by nickel-chelate affinity chromatography, and renatured by gradual removal of the denaturant. Proper isomerization of the disulfide bonds was achieved in the presence of a glutathione redox couple. After final purification by high resolution gel filtration chromatography, a monomeric form of rSPARC displaying biological activity was obtained. The recombinant protein inhibited the spreading and synthesis of DNA by endothelial cells, two properties characteristic of the native protein. We conclude that the information for the correct folding of rSPARC resides in the primary structure of the protein, and suggest that post-translational modifications are required neither for folding nor for biological activity.
Bassuk
J A
JA
Department of Biological Structure, University of Washington, Seattle 98195, USA.
Braun
L P
LP
Motamed
K
K
Baneyx
F
F
Sage
E H
EH
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007536:Isomerism; D008970:Molecular Weight; D015676:Osteonectin / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D004220:Disulfides; D015676:Osteonectin; D011994:Recombinant Proteins; D005978:Glutathione; D019803:Glutathione Disulfide