Effects of second-codon mutations on expression of the insulin-like growth factor-II-encoding gene in Escherichia coli.
Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli. A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0-22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp-IGFII in E. coli. Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.
Cantrell
A S
AS
Department of Molecular Biology, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285.
Burgett
S G
SG
Cook
J A
JA
Smith
M C
MC
Hsiung
H M
HM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic
D003062:Codon; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II