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Received: 2005-06-03

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Journal of Zhejiang University SCIENCE B 2006 Vol.7 No.1 P.13~19

http://doi.org/10.1631/jzus.2006.B0013


Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system


Author(s):  Gong Xing-guo, Ji Jing, Xie Jie, Zhou Yuan, Zhang Jun-yan, Zhong Wen-tao

Affiliation(s):  Institute of Biomacromolecule and Enzyme Engineering, School of Life Sciences, Zhejiang University, Hangzhou 310027, China

Corresponding email(s):   gongxg@cls.zju.edu.cn

Key Words:  v-Src, GST-fusion, Inclusion body, Orthogonalization, Protein tyrosine kinase


Gong Xing-guo, Ji Jing, Xie Jie, Zhou Yuan, Zhang Jun-yan, Zhong Wen-tao. Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system[J]. Journal of Zhejiang University Science B, 2006, 7(1): 13~19.

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author="Gong Xing-guo, Ji Jing, Xie Jie, Zhou Yuan, Zhang Jun-yan, Zhong Wen-tao",
journal="Journal of Zhejiang University Science B",
volume="7",
number="1",
pages="13~19",
year="2006",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.2006.B0013"
}

%0 Journal Article
%T Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system
%A Gong Xing-guo
%A Ji Jing
%A Xie Jie
%A Zhou Yuan
%A Zhang Jun-yan
%A Zhong Wen-tao
%J Journal of Zhejiang University SCIENCE B
%V 7
%N 1
%P 13~19
%@ 1673-1581
%D 2006
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.2006.B0013

TY - JOUR
T1 - Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system
A1 - Gong Xing-guo
A1 - Ji Jing
A1 - Xie Jie
A1 - Zhou Yuan
A1 - Zhang Jun-yan
A1 - Zhong Wen-tao
J0 - Journal of Zhejiang University Science B
VL - 7
IS - 1
SP - 13
EP - 19
%@ 1673-1581
Y1 - 2006
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.2006.B0013


Abstract: 
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 β-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.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1] Aviziente, E., Wyke, A.W., Jones, R.J., McLean, G.W., Westhoff, M.A., Brunton, V.G., Frame, M.C., 2002. Src-induced deregulation of E-cadher in colon cancer cells requires integrin signaling. Nat. Cell Biol., 4(8):632-638.

[2] Azuma, K., Ariki, M., Miyauchi, T., Usui, H., Takeda, M., Semba, K., Matsuzawa, Y., Yamamoto, T., Toyoshima, K., 1991. Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-Yes product, p60c-Yes. J. Biol. Chem., 266(8):4831-4839.

[3] Biscardi, J.S., Ishizawar, R.C., Parsons, S.J., 2002. Tyrosine kinase signaling in breast cancer: epidermal growth factor receptor and c-Src interactions in breast cancer. Breast Cancer Res., 2(3):203-210.

[4] Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 7(72):248-254.

[5] Brown, M.T., Cooper, J.A., 1996. Regulation, substrates and functions of Src. Biochim. Biophys. Acta, 1287(2-3):121-149.

[6] Budde, R.J., Ramdas, L., Ke, S., 1993. Recombinant pp60c-src from baculovirus-infected insect cells: purification and characterization. Prep. Biochem., 23(4):493-515.

[7] Dehm, S., Senger, M.A., Bonham, K., 2001. Src transcriptional activation in a subset of human colon cancer cell lines. FEBS Lett., 487(3):367-371.

[8] Fincham, V., Frame, M., Haefner, B., Unlu, M., Wyke, A., Wyke, J., 1994. Functions of the v-Src protein tyrosine kinase. Cell Biology International, 18(5):337-344.

[9] Frame, M.C., 2002. Src in cancer: deregulation and consequences for cell behavior. Biochem. Biophys. Acta, 1602(2):114-130.

[10] Gilmer, T.M., Erikson, R.L., 1981. Rous sarcoma virus transforming protein, p60Src, expressed in E. coli, functions as a protein kinase. Nature, 294(24):771-773.

[11] Hideyuki, S., Lee, P.S.Y., Toru, N., Izawa, I., Nakajima, M., Gallick, G.E., Levin, V.A., 1993. Bacterial expression of an active tyrosine kinase from a protein A/truncated c-Src fusion protein. FEBS Lett., 327(2):224-230.

[12] Irby, R.B., Yeatman, T.J., 2002. Increased Src activity disrupts cadherin/catenin-mediated homotypic adhesion in human colon cancer and transformed rodent cells. Cancer Res., 62(9):2669-2674.

[13] Jones, R.J., Brunton, V.G., Frame, M.C., 2000. Adhesion-linked kinases in cancer: emphasis on src, focal adhesion kinase and PI 3-kinase. European Journal of Cancer, 36(pt 13):1595-1606.

[14] Korade, M.Z., Corey, S.J., 2000. Src kinase-mediated signaling in leukocytes. J. Leukoc. Biol., 68(5):603-613.

[15] Lydon, N.B., Gay, B., Mett, H., Murray, B., Liebetanz, J., Gutzwiller, A., Piwnica-Worms, H., Roberts, T.M., McGlynn, E., 1992. Purification and biochemical characterization of non-myristoylated recombinant pp60c-Src kinase. Biochem. J., 287(pt 3):985-993.

[16] Ma, Y.C., Huang, X.Y., 2002. Novel regulation and function of Src tyrosine kinase. Cell Mol. Life Sci., 59(3):456-462.

[17] Margaret, C.F., 2002. Src in cancer: deregulation and consequences for cell behaviour. Biochimica. et Biophysica. Acta, 1602(1):114-130.

[18] Margaret, C.F., Valerie, J.F., Neil, O.C., 2002. v-Src’s hold over actin and cell adhesions. Nature Reviews Molecular Cell Biology, 3(1):233-245.

[19] Martin, G.S., 2001. The hunting of the Src. Nature Reviews Molecular Cell Biology, 2(6):467-475.

[20] Okada, M., 2000. Src signaling and its regulation. Tanpalushitsu Kakusan Koso, 45(16):2624-2631.

[21] Ptasznik, A., 2000. Crosstalk between G protein-coupled receptors and tyrosine kinase signaling: Src take center stage. Arch. Immunol. Ther. Exp., 48(1):27-30.

[22] Ram, P.T., Lyengarm, R., 2001. G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation. Oncogene, 20(13):1601-1606.

[23] Sambrook, J., Russell, D.W., 2002. Molecular Cloning: A Laboratory Manual, 3rd Ed. Science Press of China, Beijing, China, p.1723-1726 (in Chinese).

[24] Schlessinger, J., 2000. New roles for Src kinases in control of cell survival and angiogenesis. Cell, 100(3):293-296.

[25] Smith, D.B., Johnson, K.S., 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 67(1):31-40.

[26] Sun, G., Budde, R.J., 1997. Expression, purification, and initial characterization of human Yes protein tyrosine kinase from a bacterial expression system. Arch. Biochem. Biophys., 345(1):135-142.

[27] Superti-Furga, G., Courtneidge, S.A., 1995. Structure-function relationships in Src family and related protein tyrosine kinases. Bioassays, 17(4):321-330.

[28] Susa, M., Luong-Nguyen, N.H., Crespo, J., Maier, R., Missbach, M., McMaster, G., 2000. Active recombinant human tyrosine kinase c-Yes: expression in baculovirus system, purification, comparison to c-Src, and inhibition by a c-Src inhibitor. Protein Expr. Purif., 19(1):99-106.

[29] Susva, M., Missbach, M., Green, J., 2000. Src inhibitors: drugs for the treatment of osteoporosis, cancer or both? Trends in Pharmacological Sciences, 21(12):489-495.

[30] Zoller, K.E., MacNeil, I.A., Brugge, J.S., 1997. Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction. J. Immunol., 158(4):1650-1659.

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