EXPRESSION OF BIOLOGICALLY-ACTIVE HUMAN SPARC IN ESCHERICHIA-COLI

Human SPARC has been cloned by the polymerase chain reaction from an e ndothelial cell cDNA library and expressed in Escherichia coli as a bi ologically active protein. Transcriptional expression of the insert cD NA was dependent on the activation of the T7 RNA polymerase promoter b y isopropylgalactopyranoside. Two forms of recombinant SPARC (rSPARC) protein were recovered from BL21(DE3) E. coli after transformation wit h the plasmid pSPARCwt: a soluble, monomeric form of rSPARC and an ins oluble, aggregated form sequestered in inclusion bodies. The isolation of the soluble form of rSPARC was accomplished by anion-exchange, nic kel-chelate affinity, and gel filtration chromatographies. The isolate d protein was an intact, full-length polypeptide of 293 amino acids by the following criteria: N-terminal amino acid sequence, reaction with anti-SPARC immunoglobulins specific for N-terminal and C-terminal seq uences, and interaction of the C-terminal histidine tag of rSPARC with a nickel-chelate affinity resin. Circular dichroism and intrinsic flu orescence spectroscopy indicated that the conformation of rSPARC was d ependent on interaction with Ca2+ ions. The recombinant protein inhibi ted cell spreading and bound specifically to bovine aortic endothelial cells, Levels of bacterial endotoxin (<18 pg/mu g rSPARC) present in rSPARC preparations were below the threshold that affects the behavior of these endothelial cells. These conformational and biological prope rties of rSPARC are consistent with previously described characteristi cs of the native protein. The purification of biologically active rSPA RC, as well as mutated forms of the protein, will provide sufficient q uantities of protein for the determination of structure/function relat ionships. (C) 1996 Academic Press, Inc.