Alteration of amino-terminal codons of human granulocyte-colony-stimulating factor increases expression levels and allows efficient processing by methionine aminopeptidase in Escherichia coli.
We have improved the expression of recombinant human granulocyte-colony-stimulating factor (G-CSF), produced by either pL or trpP expression vectors in Escherichia coli, by altering the sequence at the 5' end of the G-CSF-coding region. Initial attempts to express G-CSF resulted in neither detectable G-CSF mRNA nor protein in the trpP system, and only G-CSF mRNA was detectable in the pL system. We modified both expression vectors to decrease the G + C content of the 5' end of the coding region without altering the predicted amino acid sequence. This resulted in expression of detectable G-CSF mRNA and protein in both systems. Expression reached 17% and 6.5% of the total soluble cellular protein in the pL and trpP expression systems, respectively. The N-terminal sequence of the recombinant G-CSF from the pL system was Met-Thr-Pro-Leu-Gly-Pro-. G-CSF isolated from several human cell lines (including the LD-1 cell line reported here), does not have an N-terminal methionyl residue. Deletion of the threonine codon at the beginning of the coding region for the mature G-CSF resulted in efficient removal of the N-terminal methionine residue during expression in E. coli.
Devlin
P E
PE
Department of Molecular Biology, Cetus Corporation, Emeryville, CA 94608.
Drummond
R J
RJ
Toy
P
P
Mark
D F
DF
Watt
K W
KW
Devlin
J J
JJ
D000626:Aminopeptidases / Q000378:metabolism*; D001483:Base Sequence; D003062:Codon; D003115:Colony-Stimulating Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014158:Transcription, Genetic
D003062:Codon; D003115:Colony-Stimulating Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D004247:DNA; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases