Mature apolipoprotein AI and its precursor proApoAI: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia coli.
Apolipoprotein AI (ApoAI) plays a central role in the regulation of lipid metabolism. Initial attempts to express human apoAI cDNA in Escherichia coli did not yield detectable levels of the mature protein. By analyzing the efficiency of expression of apoAI-lacZ gene fusions, we have been able to show that the sequence at the 5' end of the ApoAI-coding region is a critical parameter. Indeed, silent changes in the codons for the first 8 residues of ApoAI, which did not alter the amino acid sequence, affected expression dramatically. Analysis of the corresponding mRNA steady-state levels suggested a role for differential mRNA stability in the control of apoAI expression in this system. Among all the possible alternative sequences, we have identified an optimal sequence which, when reinserted in the original expression plasmid, yields high level production of mature ApoAI. This procedure has been extended to the production of the natural variant ApoAI-Milano and the precursor proApoAI. Availability of these recombinant molecules would allow the investigation of their structural and biological features. In addition, the methodology used to optimize ApoAI expression is of general interest in assuring high expression of heterologous proteins in E. coli.
Isacchi
A
A
Department of Biotechnology, Farmitalia Carlo Erba, Milano, Italy.
Sarmientos
P
P
Lorenzetti
R
R
Soria
M
M
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins