Saturation Mutagenesis of Human Interleukin-3 (∗)

A deletion variant of human interleukin-3, hIL-315-125, was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-315-125...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 270; no. 40; pp. 23754 - 23760
Main Authors:	Olins, Peter O., Bauer, S. Christopher, Braford-Goldberg, Sarah, Sterbenz, Kris, Polazzi, Joseph O., Caparon, Maire H., Klein, Barbara K., Easton, Alan M., Paik, Kumnan, Klover, Jon A., Thiele, Barrett R., McKearn, John P.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 06-10-1995 American Society for Biochemistry and Molecular Biology
Subjects:	Amino Acid Sequence Base Sequence Cell Division - drug effects Cell Line DNA Primers - genetics Escherichia coli Escherichia coli - genetics Humans Interleukin-3 - chemistry Interleukin-3 - genetics Interleukin-3 - pharmacology Molecular Sequence Data Molecular Structure Mutagenesis Mutagenesis, Site-Directed Point Mutation Protein Structure, Secondary Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - pharmacology Sequence Deletion Structure-Activity Relationship
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Summary:	A deletion variant of human interleukin-3, hIL-315-125, was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-315-125. Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-315-125 could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.270.40.23754