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A cDNA clone containing the complete human alpha 1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the alpha 1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage lambda (PL) and initiation of translation at the lambda cII 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 alpha 1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.

Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.

The primary function of alpha 1-antitrypsin (alpha 1-AT), an antiprotease produced by the liver, is the inhibition of neutrophil elastase, a protease capable of hydrolysing most connective tissue components. The importance of alpha 1-AT is demonstrated by the high incidence of early-onset emphysema in individuals with hereditary alpha 1-AT deficiency (Type PiZZ), in whom serum levels of alpha 1-AT are 10-20% of normal. Oxidants in tobacco smoke can inactivate alpha 1-AT in vitro, and studies have shown that alpha 1-AT from the lungs of individuals who smoke cigarettes may also be partially inactivated, perhaps explaining the high incidence of emphysema associated with cigarette smoking. Oxidative inactivation is probably due to modification of the Met residue (Met358) at the P1 subsite position of the elastase binding site of the protein. To study the possibility of modulating the biological properties of alpha 1-AT, we have introduced selected sequence modifications at the reactive site by in vitro mutation of a cloned alpha 1-AT complementary DNA. We describe here the characterization of two alpha 1-AT analogues produced in Escherichia coli. The first, alpha 1-AT(Met385----Val), is not only fully active as an elastase inhibitor but is also resistant to oxidative inactivation. The other, alpha 1-AT(Met358----Arg), no longer inhibits elastase but is an efficient thrombin inhibitor. The active site of the latter is identical to that of the alpha 1-AT (Pittsburgh) variant, which was associated with a fatal bleeding disorder.

Analysis of a series of lambda cII::alpha 1-antitrypsin (alpha 1AT) gene fusions of different sizes showed that increased alpha 1AT expression correlated with the stabilisation of a particular computer-predicted RNA secondary structure. Moreover, significant synthesis of unfused alpha 1AT was achieved by reconstruction of this conformation to permit interaction between the upstream region of the ribosome-binding site and the first part of the alpha 1AT coding sequence. This high-level expression was dependent upon certain silent point mutations in the coding sequence, indicating that RNA primary and secondary structure determinants can operate in concert to dictate the efficiency of protein synthesis.

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.

alpha 1-antitrypsin (alpha 1AT) deficiency is an inherited disorder almost always associated with the development of panacinar emphysema in the fourth to fifth decades. One source of alpha 1AT for chronic replacement therapy of such individuals is that produced by E.coli directed by a cDNA coding for the human alpha 1AT molecule. Using TG1(E.coli), an alpha 1AT molecule produced by E.coli transformed with the plasmid-expressing vector pTG922, the present study shows that recombinant DNA-directed E.coli-produced alpha 1AT is as an effective inhibitor of neutrophil elastase as alpha 1AT purified from plasma. Importantly, TG1(E.coli) inhibited human neutrophil elastase with an association rate constant of 1.3 +/- 0.4X10(7) M-1 sec-1, similar to that of normal plasma alpha 1AT (1.1 +/- 0.1, p greater than 0.2). Furthermore, when TG1(E.coli) was added to alpha 1AT-deficient plasma obtained from homozygous alpha 1AT type Z individuals, the TG1(E.coli) remained functional and augmented the anti-neutrophil elastase activity of the serum proportional to the amount of TG1(E.coli) added. These observations suggest that if sufficient amounts of recombinant DNA methodology-produced alpha 1AT molecules could be safely delivered to the alveolar structures of alpha 1AT-deficient individuals, they would function to protect the alveolar walls from elastolytic attack.

Seven active site variants of human alpha 1-antitrypsin (alpha 1AT) were produced in Escherichia coli following site-specific mutagenesis of the alpha 1AT complementary DNA. alpha 1AT (Ala358), alpha 1AT (Ile358) and alpha 1AT (Val358) were efficient inhibitors of both neutrophil and pancreatic elastases, but not of cathepsin G. alpha 1AT (Ala356, Val358) and alpha 1AT (Phe358) specifically inhibited pancreatic elastase and cathepsin G respectively. The most potent inhibitor of neutrophil elastase was alpha 1AT (Leu358), which also proved to be effective against cathepsin G. The alpha 1AT (Arg358) variant inactivated thrombin with kinetics similar to antithrombin III in the presence of heparin. Electrophoretic analysis showed that SDS-stable high mol. wt complexes were formed between the mutant inhibitors and the cognate proteases in each case. These data indicate that effective inhibition occurs when the alpha 1AT P1 residue (position 358) corresponds to the primary specificity of the target protease. Moreover, alteration of the P3 residue (position 356) can further modify the reactivity of the inhibitor. Two of the variants have therapeutic potential: alpha 1AT (Leu358) may be more useful than plasma alpha 1AT in the treatment of destructive lung disorders and alpha 1AT (Arg358) could be effective in the control of thrombosis.