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 1995-10, Vol.270 (40), p.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: Article
Language:English
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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.270.40.23754