Selection of mutations that increase alpha 1-antitrypsin gene expression in Escherichia coli.
The gene encoding human alpha-1-antitrypsin (A1AT), when cloned and expressed as a full-length, non-fusion gene product in Escherichia coli, accumulates to levels up to 0.1% of total cellular protein. Truncation of the gene at its 5' end or synthesis as a fusion protein increases expression up to 200-fold. Extensive mutagenesis in vitro within this same 5'-terminal region aimed at improving codon usage and disrupting potential secondary structure increased expression only 10 to 20-fold. We have developed a translational fusion system for selecting mutations and applied it to the study of A1AT expression in E. coli. With this methodology, we have obtained single base-pair mutations having up to a 20-fold effect on A1AT expression. When we combined these multiple single base-pair mutations, we achieve up to a 200-fold increase in A1AT expression. The resulting gene product is of authentic size (394 amino acid residues) and contains two amino acid substitutions (Asn in place of Asp) in codons 2 and 6. This protein is primarily in the soluble fraction of the E. coli lysate and has identical activity to A1AT purified from human sera. The methodology used to generate these mutations may be generally applicable to the study of genes that do not express well in E. coli initially, and provides an alternative to secondary structure analysis in the redesign of such genes.
Sutiphong
J
J
Smith Kline & French Laboratories, Research and Development, King of Prussia, PA 19406-2799.
Johansen
H
H
Sathe
G
G
Rosenberg
G S
GS
Shatzman
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D014158:Transcription, Genetic; D000515:alpha 1-Antitrypsin / Q000235:genetics*
D000515:alpha 1-Antitrypsin