High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.

Abstract

A cDNA clone containing the complete human a1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the a1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage A (PL) and initiation of translation at the A cli gene ribosome-binding site. This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein. The polypeptide produced was recognized by antisera raised against human a1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.a1-Antitrypsin is a serum antiprotease of hepatic origin whose most important physiological role is to restrict neutrophil elastase activity in the lung (1-2). A deficiency of a1-antitrypsin upsets the alveolar protease-antiprotease balance, leading to elastase-mediated tissue destruction and chronic pulmonary emphysema (3). Inherited a1-antitrypsin deficiency occurs at a high frequency in European populations (1 in 750 for the two principal variants Z and S) (4). The most common clinically significant variant (type Z) has a single amino acid substitution that is associated with reduced glycosylation of the a1-antitrypsin molecule (5-6). This results in its accumulation in hepatocytes and a reduction in serum concentration to 10-15% of normal (7). Cigarette smoking, a major factor in nonhereditary emphysema, also causes a protease-antiprotease imbalance in the lower respiratory tract (8) as a consequence of both increased elastase levels and a 50% reduction in active alveolar a1-antitrypsin (9)(10). Clinical trials have shown that a1-antitrypsin deficiency can be treated by replacement therapy (11), but the problems of possible viral contamination associated with the use of human blood products deter extensive clinical use of a1-antitrypsin purified from serum. To circumvent this problem we have used the techniques of genetic engineering to produce human a1-antitrypsin in a microorganism. The availability of information on the sequences of baboon and human a1-antitrypsin cDNA clones (12, 13) and the structures of normal and variant genes (14, 15) enabled us to isolate a full-length human a1-antitrypsin cDNA clone. Transfer of this sequence into a high-level expression system resulted in a recombinant E. coli strain capable of synthesizing a1-antitrypsin at levels of up to 15% of total cell protein. MATERIALS AND METHODSBacterial Strains and Plasmids. cDNA banks were prepared using E. coli strain 1106 (supE hsdS met supF). Strain TGE900 [F-su-ilv-bio (XcI857ABamAHI)], which produces the temperature-sensitive XcI857 repressor, was used as host for the PL-containing plasmids.pTG603 is a pBR322 derivative containing a human a1-antitrypsin cDNA insert. pTG920 is the PL-containing expression vector and pTG922 is a derivative that expresses human a1-antitrypsin.Isolation of a1-...

The purification of eukaryotic polypeptides synthesized in Escherichia coli

“…Examples include ,J-galactosidase fusions with HBV pre-S2 region (Offensperger et al, 1985) and cholera toxin CTP3 (Jacob et al, 1985). AcII protein sequences fused to al-antitrypsin (Courtney et al, 1984) and AN protein sequences fused to HSV-thymidine kinase (Waldman et al, 1983). The latter is an interesting example of a fusion protein in which there was no specific cleavage site.…”

“…al-Antitrypsin was not completely soluble, and approx. 60% of the fusion protein was insoluble and inactive (Courtney et al, 1984).…”

Mutations in the amino‐terminal domain of the human poly(ADP‐ribose) polymerase that affect its catalytic activity but not its DNA binding capacity

“…The amplified fragment (1100 bp) bearing random mutations encompasses the HindllI site (nucleotide 21 upstream of the ATG) and the BstEII site (nucleotide 998). Following HindlII BstElI restriction, the library of mutated DBD was cloned into the same sites of the prokaryotic expression vector pTG PARP [15], now designated pTG PARP*. This vector was used directly for transformation of the E. coli TGE 900 strain [15] from which full-length PARP-bearing mutations in the DBD only (PARP*) was overproduced.…”

Effects of light stress on the expression of early light‐inducible proteins in barley

“…[17], from which the single-stranded-DNA-binding-protein gene was first removed with Ban. Since ELIP expression, which is under the control of the heat-inducible A promoter using Escherichiu coli TGE 900 [18], could not be detected in this system, the insert was removed with EcoRI and ligated into the EcoRI site of the vector pGEX2T [19]. This procedure yielded a gene coding for a fusion protein of glutathione S-transferase and a small barley ELIP precursor, under control of the tuc promoter.…”