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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-...

A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

For many patients requiring coronary reoperations, off-pump techniques are safe and feasible. Complete revascularization was achieved in at least 75% of patients in an unselected population, with mortality and perioperative event rates that are comparable with those of conventionally performed coronary reoperations.

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