In vitro genetic techniques were used to study the sequence requirements for the initiation of specific transcription. Deletion mutants were constructed around the putative promoter of the adenovirus-2 major late and chicken conalbumin genes. Specific transcription in vitro by RNA polymerase B together with a HeLa cell cytoplasmic extract was used as the test for promoter function. With this approach sequences which are essential for the initiation of specific transcription in vitro, were shown to be located between 12 and 32 base pairs upstream from the 5' end of these genes.
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 amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.
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