High-level production of fully active human alpha 1-antitrypsin in Escherichia coli.
The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein. In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein. The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents. In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT. The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons. These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region. Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein. Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants. Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application.
Johansen
H
H
Biopharmaceutical Research, Smith Kline & French Laboratories, King of Prussia, PA 19406-2799.
Sutiphong
J
J
Sathe
G
G
Jacobs
P
P
Cravador
A
A
Bollen
A
A
Rosenberg
M
M
Shatzman
A
A
D001483:Base Sequence; D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D006801:Humans; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology
D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D004262:DNA Restriction Enzymes