Abstract
We have generated random variants of human interleukin-2 (hIL-2) and assayed them using a rapid method that is generally applicable to proteins secreted by yeast. Mutein screening is performed in three steps: (1) in vitro mutagenesis of the target gene and selection of mutant genes by a physical method, (2) insertion of mutant genes into a yeast secretion vector, followed by yeast transformation, and (3) biological assay of protein variants secreted by the transformants into the culture fluid. In this way, we produced and tested about 500 variants of hIL-2 for activity; 155 variants were found to be biologically inactive. Sequence analyses of genes encoding the inactive variants revealed residues essential for hIL-2 activity, including residues at the extreme C-terminus and within a C-terminal α-helix (helix E).
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References
Knowles, J.R. 1987. Tinkering with enzymes: what are we learning? Science 236:1252–1258.
Wells, J.A. and Powers, D.B. 1986. In vivo formation and stability of engineered disulfide bonds in subtilisin. J. Biol. Chem. 261:6564–6570.
Rosenberg, S., Barr, P.J., Najarian, R.C., and Hallewell, R.A. 1984. Synthesis in yeast of a functional oxidation-resistant mutant of human alpha-antitrypsin. Nature 312:77–80.
Pantoliano, M.W., Ladner, R.C., Bryan, P.N., Rollence, M.L., Wood, J.F. and Poulos, T.L. 1987. Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond. Biochemistry 26:2077–2082.
Bryan, P.N., Rollence, M.L., Pantoliano, M.W., Wood, J., Finzel, B.C., Gilliland, G.L., Howard, A.J. and Poulos, T.L. 1986. Proteases of enhanced stability: characterization of a thermostable variant of subtilisin. Proteins 1:326–334.
Marcucci, F. and De Maeyer, E. 1986. An interferon analogue, [Ala30,32,33]HuIFN-α2, acting as a HuIFN-α2 antagonist on bovine cells. Biochim. Biophys. Res. Commun. 134:1412–1418.
Wang, A., Lu, S.-D. and Mark, D.F. 1984. Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science 224:1431–1433.
Myers, R.M., Lerman, L.S. and Maniatis, T. 1985. A general method for saturation mutagenesis of cloned DNA fragments. Science 229:242–247.
Smith, K.A. 1988. lnterleukin-2: Inception, impact, and implications. Science 240:1169–1176.
Liang, S-M., Thatcher, D.R., Liang, C-M. and Allet, B. 1986. Studies of structure-activity relationships of human interleukin-2. J. Biol. Chem. 261:334–337.
Zurawski, S.M., Mosmann, T.R., Benedik, M. and Zurawski, G. 1986. Alterations in the ammo-terminal third of mouse interleukin 2: effects on biological activity and immunoreactivity. J Immunol. 137:3354–3360.
Cohen, F.E., Kosen, P.A., Kuntz, I.D., Epstein, L.B., Ciardelli, T.L. and Smith, K.A. 1986. Strucutre-activity studies of interleukin-2. Science 234:349–352.
Miyaji, H., Nishi, T., Saito, A., Maeda, S., Shimada, K., Hirano, T., Onouc, K. and Itoh, S. 1987. Expression of mature human interleukin 2 and its derivatives in Escherichia coli and comparison of their biological activity in vitro. Agric. Biol. Chem. 51:1135–1142.
Ju, G., Collins, L., Kaffka, K.L., Tsien, W-H., T., Chizzonite, R., Crowl, R., Bhatt, R. and Kilian, P.L. 1987. Structure-function analysis of human interleukin-2. J. Biol. Chem. 262:5723–5731.
Ciardelli, T.L., Landgraf, B., Gadski, R., Strnad, J., Cohen, F.E. and Smith, K.A. 1988. A design approach to the structural analysis of interleukin-2. J. Mol. Recog. 1:42–47.
Weir, M.P., Chaplin, M.A., Wallace, D.M., Dykes, C.W. and Hobden, A.N. 1988. Structure-activity relationships of recombinant human interleukin 2. Biochemistry 27:6883–6892.
Zurawski, S.M. and Zurawski, G. 1988. Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis. EMBO J. 7:1061–1069.
Brandhuber, B.J., Boone, T., Kenney, W.C. and McKay, D.B. 1987. Three-dimensional structure of interleukin-2. Science 238:1707–1709.
Barr, P.J., Bleackley, R.C., Brake, A.J. and Merryweather, J.P. 1984. Yeast alpha-factor directed secretion of human interleukin-2 from a chemically synthesized gene. J Cell Biochem. Suppl. 8A:23
Ernst, J.F. 1986. Improved secretion of heterologous proteins by Saccharomyces cerevisiae: effects of promoter substitution in alpha-factor fusions. DNA 5:483–491.
Browning, J. and Mattaliano, R. 1986. Biologically active proteolytic fragments of interleukin-2. J. Cell. Biochem. Suppl. 10A:73.
Mosmann, T. 1983. Rapid colorimetic assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55–63.
Reidhaar-Olson, J.F. and Sauer, R.T. 1988. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241:53–57.
Ernst, J.F., Hampsey, D.M., Stewart, J.W., Rackovsky, S., Goldstein, D. and Sherman, F. 1985. Substitutions of proline 76 in yeast iso-1-cytochrome c. J. Biol. Chem. 260:13225–13236.
Kernpenaers, W. 1984. Ph.D. thesis. University of Gent, Belgium.
Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Ito, H., Fukuda, Y., Murata, K. and Kimura, A. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168.
Sherman, F., Fink, G. and Hicks, J. 1981. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Levison, A., Silver, D. and Seed, B. 1984. Minimal size plasmids containing an M13 origin for production of single strand transducing particles. J. Mol. Appl. Genet. 2:507–517.
Towbin, A., Staehlin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. 76:4350–4354.
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Ernst, J., Richman, L. Screening of Muteins Secreted by Yeast: Random Mutagenesis of Human Interleukin-2. Nat Biotechnol 7, 716–720 (1989). https://doi.org/10.1038/nbt0789-716
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DOI: https://doi.org/10.1038/nbt0789-716