Isolation and properties of recombinant DNA produced variants of human alpha 1-proteinase inhibitor.

Although it is widely recognized that many proteins contain discrete functional domains, it is less certain whether smaller, less obviously discrete, units of structure will retain their specific function when transplanted into a different context. The observation that the potent inflammatory cytokine human interleukin 1 beta has the same overall structure as soybean trypsin inhibitor (STI) (Kunitz) prompted us to replace a tight turn in the cytokine sequence with the large loop in soybean trypsin inhibitor that binds to the active site of trypsin. Wild-type interleukin 1 beta (IL-1 beta) is highly resistant to proteolysis, but the chimeric STI/IL is specifically cleaved by trypsin, apparently in the inserted loop. Other chimeric interleukins have also been constructed, by replacing the same tight turn with inhibitory loops from other protein protease inhibitors: turkey ovomucoid inhibitor (TOI), a chymotrypsin inhibitor, and alpha 1-antitrypsin (AT), an elastase inhibitor. Although these loops come from proteins not related structurally to interleukin 1, they confer specific protease sensitivity or inhibition on the chimeric cytokine. The cytokine properties of these chimeric interleukins have also been evaluated. The chimeras formed from human IL-1 beta and all inhibitory loops tested bind to the interleukin 1 receptor with reasonable affinity. The typical cellular effects of IL-1, however, are not observed with all the recombinant proteins, thus confirming that receptor binding and signal transduction can be uncoupled. When these results are taken together with the results of site-directed mutagenesis of IL-1, reported in this paper and elsewhere, they allow the receptor and intracellular transduction sites on the protein to be mapped in detail.

Section: Methodsmentioning

confidence: 99%

Modularity of protein function: Chimeric interleukin 1.beta.s containing specific protease inhibitor loops retain function of both molecules

Wolfson¹,

Kanaoka²,

Lau³

et al. 1993

“…Materials and Methods Materials. Yeast 2u plasmid PC1/1 (Travis et al, 1985) and shuttle vector pBSlOO (Barr et al, 1987) were gifts from Dr. P. Barr (Chiron Corp.). Expression vector pAR3038 and Escherichia coli K12 strain BL21 (Studier & Moffat, 1986) were gifts from Dr. W. Studier (Brookhaven National Laboratories).…”

mentioning

confidence: 99%

“…pEl (pKK223.3) was cloned into JM105 (lacIQ); pE2 (pAR3038) was originally cloned and propagated in HB101 and subsequently cloned into expression host BL21 (Studier & Moffat, 1986); pE3a and pE3b (pMG27N-S and pOTS-Ncol), originally cloned and expanded in MM294cI+, were cloned into expression hosts N5151 and AR120, respectively (Shatzman & Rosenberg, 1986). Yeast expression vectors pE4a and pE4b (PCl/l; Travis et al, 1985) were originally cloned and propagated in E. coli HB101 and then transformed into S. cerevisiae strain AB103.1 (Beggs, 1978). Growth Conditions.…”

mentioning

confidence: 99%

Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major

Grumont

Sirawaraporn²,

Santi³

1988

The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) of Leishmania major has been cloned and expressed in Escherichia coli and Saccharomyces cerevisiae. The strategy involved placing the entire 1560-bp coding sequence into a parent cloning plasmid that was designed to permit introduction of unique restriction sites at the 5'- and 3'-ends. In this manner, the entire coding sequence could be easily subcloned into a variety of expression vectors. High levels of TS-DHFR gene expression were driven by tac, pL and T7 RNA pol promoters in E. coli, and the GAPDH-ADH-2 promoter in S. cerevisiae. L. major TS-DHFR also complemented TS deficiency in E. coli. In E. coli, the protein accumulated to very high levels, but most was present as inactive inclusion bodies. Nevertheless, substantial amounts were soluble; up to 2% of the soluble protein was catalytically active TS-DHFR. In the yeast systems, essentially all of the bifunctional protein was soluble and catalytically active, and crude extracts contained about 100-fold more enzyme than do extracts from wild-type L. major. The expressed TS-DHFR from yeast and E. coli was purified to homogeneity by methotrexate-Sepharose affinity chromatography. About 8.5 mg of homogeneous, catalytically active protein is obtained from a 1-L culture of yeast, and 1.5 mg was obtained from 1 L of E. coli culture. A 200-L fermentation of the yeast expression system yielded a crude extract containing over 4 g of TS-DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)

“…a-l-PI is an elastase inhibitor that is uniquely sensitive to oxidants (Travis & Salvesen, 1983) and is currently under intense investigation because of its potential role in the pathogenesis of lung and joint disease (Jeppsson & Laurell, 1 Abbreviations: PA, plasminogen activator; PAI, plasminogen activator inhibitor; tPA, tissue-type plasminogen activator; BAEs, bovine aortic endothelial cells; MEM, minimal essential medium; PBS, phosphate-buffered saline (0.14 M NaCl/0.01 M sodium phosphate, pH 7.2); SDS, sodium dodecyl sulfate; TX-100, Triton X-100; PAGE, polyacrylamide gel electrophoresis; RFA, reverse fibrin autography; DDT, dithiothreitol; a-l-PI, arprotease inhibitor; BSA, bovine serum albumin; CM, conditioned medium; Serpin, serine protease inhibitor; 1975; Travis & Salveson, 1983). The inactivation of a-l-PI by oxidants results from the conversion of a single methionine residue in the exposed reactive site of the molecule into methionine sulfoxide (Johnson & Travis, 1978, 1979Abrams et al, 1981;Travis & Salveson, 1983;Travis et al, 1985). The data presented here suggest that a similar mechanism may be responsible for the loss of PAI activity upon iodination.…”

mentioning

confidence: 60%

Inactivation of plasminogen activator inhibitor by oxidants

Lawrence¹,

Loskutoff²

1986

131

The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cysteine residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. The PAI was more sensitive to oxidative inactivation than urokinase, elastase, and alpha 1-protease inhibitor. Incubation of the chloramine T inactivated PAI with methionine sulfoxide peptide reductase in the presence of dithiothreitol (DTT) restored more than 90% of the PAI activity. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles alpha 1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.