Functional expression of bitistatin, a disintegrin with potential use in molecular imaging of thromboembolic disease.
Bitistatin is a single-chain disintegrin which contains 83 amino acids and is internally crosslinked with seven disulfide bonds. This platelet aggregation inhibitor, which binds with high affinity to the alphaIIbbeta3 integrin, has potential use as the basis for a radiotracer to locate thrombi and emboli by scintigraphic imaging. A method amenable to large-scale, consistent production of bitistatin was sought. A synthetic gene coding for bitistatin was inserted into two different Escherichia coli expression vectors. One vector expressed recombinant bitistatin (rBitistatin) as a cleavable fusion protein and the other expressed rBitistatin as an isolated protein. In both cases, rBitistatin contained an additional amino acid (Gly) at the N-terminus compared with the native protein. The fusion protein was purified by affinity chromatography, then cleaved enzymatically to release rBitistatin, which was purified by reversed-phase high performance liquid chromatography (HPLC) to a single active form. The rBitistatin produced as an isolated protein was purified from cell lysate by HPLC in a reduced form, then refolded, and purified again by HPLC. Yields of active rBitistatin averaged 12 mg/L for expression as an isolated protein, 10 times as high as when the fusion protein was employed. Structural assays confirmed the expected mass and sequence of the product. Functional assays (inhibition of platelet aggregation in vitro, equilibrium binding to platelets in vitro, and binding of labeled protein to experimental thrombi and emboli in vivo) confirmed that rBitistatin retained the functional characteristics of native bitistatin.
Knight
Linda C
LC
Nuclear Medicine Division, Radiology Department, Temple University School of Medicine, Philadelphia, PA 19140, USA. Lknight@temple.edu
Romano
Jan E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010957:Plasmids; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D020673:Protein Renaturation; D011877:Radionuclide Imaging; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012910:Snake Venoms; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013923:Thromboembolism / Q000000981:diagnostic imaging* / Q000378:metabolism
D003432:Cross-Linking Reagents; D004220:Disulfides; D007457:Iodine Radioisotopes; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D012910:Snake Venoms; C062186:bitistatin; D005982:Glutathione Transferase; D005998:Glycine