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 ost eonectin 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 funda mental role for this protein in tissue morphogenesis and cellular diff erentiation. Human SPARC was cloned by the polymerase chain reaction f rom 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 ch romatography, and renatured by gradual removal of the denaturant. Prop er isomerization of the disulfide bonds was achieved in the presence o f a glutathione redox couple. After final purification by high resolut ion gel filtration chromatography, a monomeric form of rSPARC displayi ng biological activity was obtained. The recombinant protein inhibited the spreading and synthesis of DNA by endothelial cells, two properti es characteristic of the native protein. We conclude that the informat ion 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. Copyright ( C) 1996 Published by Elsevier Science Ltd