Optimized bacterial expression of human apolipoprotein A-I.
Apolipoprotein A-I (apoA-I) serves critical functions in plasma lipoprotein metabolism as a structural component of high density lipoprotein, activator of lecithin:cholesterol acyltransferase, and acceptor of cellular cholesterol as part of the reverse cholesterol transport pathway. In an effort to facilitate structure:function studies of human apoA-I, we have optimized a plasmid vector for production of recombinant wild type (WT) and mutant apoA-I in bacteria. To facilitate mutagenesis studies, subcloning, and DNA manipulation, numerous silent mutations have been introduced into the apoA-I cDNA, generating 13 unique restriction endonuclease sites. The coding sequence for human apoA-I has been modified by the introduction of additional silent mutations that eliminate 18 separate codons that employ tRNAs that are of low or moderate abundance in Escherichia coli. Yields of recombinant apoA-I achieved using the optimized cDNA were 100+/-20 mg/L bacterial culture, more than fivefold greater than yields routinely obtained with the original cDNA. Site-directed mutagenesis of the apoA-I cDNA was performed to generate a Glu2Asp mutation in the N-terminal sequence of apoA-I. This modification, which creates an acid labile Asp-Pro peptide bond between amino acids 2 and 3, permits specific chemical cleavage of an N-terminal His-Tag fusion peptide used for rapid protein purification. The product protein's primary structure is identical to WT apoA-I in all other respects. Together, these changes in apoA-I cDNA and bacterial expression protocol significantly improve the yield of apoA-I protein without compromising the relative ease of purification.
Ryan
Robert O
RO
Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
Forte
Trudy M
TM
Oda
Michael N
MN
D016632:Apolipoprotein A-I / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005561:Formates / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D016632:Apolipoprotein A-I; D003062:Codon; D005561:Formates; D011994:Recombinant Proteins; C030544:formic acid; D004262:DNA Restriction Enzymes