RESUMEN
v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl beta-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.
Asunto(s)
Proteínas Bacterianas , Química , Genética , Glutatión Transferasa , Genética , Proteína Oncogénica pp60(v-src) , Química , Genética , Ingeniería de Proteínas , Métodos , Proteínas Recombinantes de Fusión , Química , Saccharomyces cerevisiae , Genética , MetabolismoRESUMEN
CPTP1 is a nontransmembrane chicken protein tyrosine phosphatase having 92% sequence homology to the corresponding 321 amino acids of human protein tyrosine phosphatase 1B (HPTP1B). Using anti-CPTP1 antibody, we identified CPTP1-like rat PTP1 of 51 kappa Da in Rat-1 and v-src-transformed Rat-1 fibroblasts. Here we show that CPTP1-like rat PTP1 binds to p60v-src in vivo and CPTP1 also can associate with p60v-src in cell lysate of v-src- transformed Rat-1 fibroblasts. Interaction between HPTP1B-type PTPs, CPTP1-like rat PTP1 and CPTP1, and p60v-src was reduced by vanadate treatment for 13 h due to down regulation of the protein level of p60v-src in vivo. Interestingly, CPTP1-like rat PTP1 was coimmunoprecipitated with a 70-kappa Da protein which has a possibility to be tyrosine- phosphorylated by p60v-src in v-src-transformed Rat- 1 fibroblasts. These results suggest that HPTP1B- type PTPs may play an important role in p60src dependent signal pathway in eucaryotic cells.