Saturation mutagenesis of human interleukin-3.
A deletion variant of human interleukin-3, hIL-3(15-125), 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-3(15-125). 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-3(15-125) 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.
Olins
P O
PO
Searle R & D, Monsanto Company, St. Louis, Missouri 63198, USA.
Bauer
S C
SC
Braford-Goldberg
S
S
Sterbenz
K
K
Polazzi
J O
JO
Caparon
M H
MH
Klein
B K
BK
Easton
A M
AM
Paik
K
K
Klover
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D017931:DNA Primers; D007377:Interleukin-3; D011994:Recombinant Proteins