Expression of biologically active human SPARC in Escherichia coli.
Human SPARC has been cloned by the polymerase chain reaction from an endothelial cell cDNA library and expressed in Escherichia coli as a biologically active protein. Transcriptional expression of the insert cDNA was dependent on the activation of the T7 RNA polymerase promoter by isopropylgalactopyranoside. Two forms of recombinant SPARC (rSPARC) protein were recovered from BL21 (DE3) E. coli after transformation with the plasmid pSPARCwt: a soluble, monomeric form of rSPARC and an insoluble, aggregated form sequestered in inclusion bodies. The isolation of the soluble form of rSPARC was accomplished by anion-exchange, nickel-chelate affinity, and gel filtration chromatographies. The isolated 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 sequences, and interaction of the C-terminal histidine tag of rSPARC with a nickel-chelate affinity resin. Circular dichroism and intrinsic fluorescence spectroscopy indicated that the conformation of rSPARC was dependent on interaction with Ca2- ions. The recombinant protein inhibited cell spreading and bound specifically to bovine aortic endothelial cells. Levels of bacterial endotoxin (< 18 pg/microgram rSPARC) present in rSPARC preparations were below the threshold that affects the behavior of these endothelial cells. These conformational and biological properties of rSPARC are consistent with previously described characteristics of the native protein. The purification of biologically active rSPARC, as well as mutated forms of the protein, will provide sufficient quantities of protein for the determination of structure/function relationships.
Bassuk
J A
JA
Department of Biological Structure, University of Washington, Seattle 98195, USA.
Baneyx
F
F
Vernon
R B
RB
Funk
S E
SE
Sage
E H
EH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004730:Endothelium, Vascular / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D015676:Osteonectin; D011994:Recombinant Proteins; D002118:Calcium