Molecular cloning and expresion in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-Gln and Glu73-Gln mutants

• Autorzy: Bolewska K. , Kozlowska H. , Groch G. , Mikolajek B. , Bierzynski A.
• Języki publikacji: EN

Abstrakty

EN

Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.

Słowa kluczowe

EN

cell cycle; gene expression; gene coding; mutagenesis; purification; mutant; retention time; growth; expression system; calcium; regulation; protein; Escherichia coli; recombinant protein; differentiation; gene cloning

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 1997
• Tom: 44
• Numer: 2
• Opis fizyczny: p.275-283,fig.

Twórcy

autor

Bolewska K.

• Polish Academy of Sciences, A.Pawinskiego 5a, 02-106 Warsaw, Poland

autor

Kozlowska H.

autor

Groch G.

autor

Mikolajek B.

autor

Bierzynski A.

Bibliografia

• 1. Kligman, D. & Hilt, D.C. (1988) The S100 protein family. Trends Biochem. Sci. 13, 437—443.
• 2. Van Eldik, L.J. & Zimmer, D.B. (1988) Mecha­nism of action of the S100 family of calcium modulated proteins; in Calcium and Calcium Rinding Proteins (Gerday, C., Gilles, T. & Bolis, L., eds.) pp. 114-127, Springer Verlag, Berlin.
• 3. Donato, R. (1991) Perspectives in S-100 pro­tein biology. Review article. Cell Calcium 12, 713-726.
• 4. Hilt, D.C. & Kligman, D. (1991) The S-100 protein family: A biochemical and functional overview; in Novel Calcium-Binding Proteins (Heizmann, C.W., cd.) pp. 65-103, Springer Verlag, Berlin.
• 5. Schafer, B.W. & Heizmann, C.W. (1996) The S100 family of EF-hand calcium-binding pro­teins: Functions and pathology. Trends Bio- chem. Sci. 21,134-140.
• 6. Kuwano, R., Usui, H., Maeda, T., Araki, K., Yamakuni, T.t Kurihara, T. & Takahashi, Y. (1987) Tissue distribution of rat S-lOOa and p subunits mRNAs. Mol Brain Res. 2, 79-S2.
• 7. Kato, K., Kimura, S., Haimoto, H. & Suzuki, F. (1986) SlOOao(aa) protein: Distribution in muscle tissues of various animals and purifi­cation from human pectoral muscle. J. Neuro- chem. 46, 1555-1560.
• 8. Engelkamp, D., Schafer, B.W., Erne, P. & Heizmann, C.W. (1992) S100 alpha, CAPL, and CACY: Molecular cloning and expression analysis of three calcium-binding proteins from human heart. Biochemistry 31, 10258- 10264.
• 9. Zimmcr, D.B. & Van Eldik, L.J. (1986) Iden­tification of a molecular target for the cal­cium-modulated protein S100. Fructose-1,6- bisphosphate aldolase. J. Biol. Chem. 261, 11424-11428.
• 10. Donato, R. (1988) Calcium-independent, pH- regulated effects of S-100 proteins on assem- bly-disassembly of brain microtubule protein in vitro. J. Biol. Chem. 263, 106-110.
• 11. Fano, G., Angelella, P., Mariggio, D., Aisa, M.C., Giambanco, I. & Donato, R. (1989) S- lOOao protein stimulates the basal (Mg2+-ac- tivated) adenylate cyclase activity associated with skeletal muscle membranes. FEBS Lett. 248, 9-12.
• 12. Fano, G., Marsili, V., Angelella, P., Aisa, M.C., Giambanco, I. & Donato, R. (1989) S-lOOaoa ft,protein stimulates Ca -induced Ca release from isolated sarcoplasmic reticulum vesicles. FEBS Lett. 255, 381-384.
• 13. Baudier, J., Bergeret, E., Bertacchi, N., We- intraub, H., Gagnon, J. & Garin, J. (1995) Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and SlOOa (aa) proteins. Biochemistry 34, 7834- 7846.
• 14. Baudier, J., Delphin, C., Grunwald, D., Khochbin, S. & Lawrence, J.J. (1992) Charac­terization of the tumor suppressor protein p53 as a protein kinase C substrate and a SlOOb- binding protein. Proc. Natl. Acad. Sci. U.SA. 89,11627-11631.
• 15. Zimmer, D.B. & Dubuisson, J.G. (1993) Iden­tification of an SI 00 target protein: Glycogen phosphorylase. Cell Calcium, 14, 323-332.
• 16. Isobe, T. & Okuyama, T. (1981) The amino- acid sequence of the alpha subunit in bovine brain S-lOOa protein. Eur. J. Biochem. 116, 79-86.
• 17. Boguta, G., St^pkowski, D. & Bierzynski, A. (1988) Theoretical estimation of the calcium- binding constants for proteins from the tro­ponin C superfamily based on a secondary structure prediction method. I. Estimation procedure.«/. Theor. Biol. 135, 41-61.
• 18. Carlstrom, G. & Chazin, W.J. (1993) Two-di­mensional 1H nuclear magnetic resonance studies of the half-saturated (Ca )i state of calbindin D9k- Further implications for the molecular basis of cooperative Ca binding. J. Mol. Biol. 231, 415—430.
• 19. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Man­ual]; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
• 20. Grosjean, H. & Walter, F. (1982) Preferential codon usage in prokaryotic genes: The optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18, 199-209.
• 21. Doering, D.S. (1992) Ph.D. Thesis, Massachu­setts Institute of Technology.
• 22. Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J. & Studier, F.W. (1987) Vec­tors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56, 125-135.
• 23. Weissman, J.S. & Kim, P.S. (1992) The pro region of BPTI facilitates folding. Cell 71, 841-851.
• 24. Lumb, K.J., Carr, C.M., & Kim, P.S. (1994) Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4. Biochemistry 33, 7361-7367.
• 25. Peng, Z.-yu, Wu, L.C. & Kim, P.S. (1995) Local structural preferences in the alpha-lactalbu- min molten globule. Biochemistry 34, 3248- 3252.
• 26. Bolewska, K., Krowarsch, D., Otlewski, J., Jaroszewski, Ł. & Bierzyński, A. (1995) Syn­thesis, cloning and expression in Escherichia coli of a gene coding for the Met8-»Leu CMTI I - a representative of the squash inhibitors of serine proteinases. FEBS Lett. 377, 172-174.
• 27. Kunkel, T.A., Roberts, J.D. & Zakour, R.A. (1987) Rapid and efficient site-specific mu­tagenesis without phenotypic selection. Meth­ods Enzymol. 154, 367-382.
• 28. Studicr, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. (1990) Use of T7 RNA po­lymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89.
• 29. Donato, R., Giambanco, I., Aisa, M.C., di Geronimo, G., Ceccarelli, P., Rambotti, M.G. & Spreca, A. (1989) Cardiac S-100ao protein: Purification by a simple procedure and re­lated immunocytochemical and immunoche­mical studies. Cell Calcium 10, 81-92.
• 30. Ambler, R.P. (1972) Carboxypeptidases A and B. Methods Enzymol. 25, 262-272.
• 31. Seiler, N. (1970) Use of the dansyl reaction in biochemical analysis. Methods Biochem. Anal. 18, 259-337.
• 32. Tapuhi, Y., Schmidt, D.E., Linder, W. & Karger, B.L. (1981) Dansylation of amino ac­ids for high-performance liquid chromatogra­phy analysis. Anal. Biochem. 115, 123-129.
• 33. Lukas, T.J. & Watterson, M. (1988) Purifica­tion of calmodulin and preparation of immo­bilized calmodulin. Metlwds Enzymol. 157, 328-339.
• 34. Gibbs, F.E.M., Wilkinson, M.C., Rudland, P.S. & Barraclough, R. (1994) Interactions in vitro of p9Ka, the rat S-100-related, metasta­sis-inducing, calcium-binding protein. J. Biol. Chem. 269,18992-18999.
• 35. Van Eldik, L.J., Staecker, J.L. & Winning- ham-Major, F. (1988) Synthesis and expres­sion of a gene coding for the calcium-modu­lated protein S100 beta and designed for cas­sette-based, site-directed mutagenesis. ,J. Biol. Chem. 263, 7830-7837.
• 36. Pedrocchi, M., Schafer, B.W., Durussel, I., Cox, JA. & Heizmann, C.W. (1994) Purifica­tion and characterization of the recombinant human calcium-binding S100 proteins CAPL and CACY. Biochemistry 33, 6732-6738.
• 37. Fohr, U.G., Heizmann, C.W., Engelkamp, D., Schafer, B.W. & Cox, J.A. (1995) Purification and cation binding properties of the recombi­nant human S100 calcium-binding protein A3, an EF-hand motif protein with high affin­ity for zinc. J. Biol. Chem. 270, 21056-21061.
• 38. Wetzel, R., Perry, L.J., Estell, D.A., Lin, N., Levine, H.L., Slinker, B., Fields, F., Ross, M.J. & Shively, J. (1981) Properties of a human alpha-interferon purified from E. coli ex­tracts. J. Interferon Res. 1, 381-390.