Expression of human plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli as a soluble protein comprised of active and latent forms. Isolation and crystallization of latent PAI-1.

Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of 'latent' PAI-1. Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2-376 of the 379 amino acid serpin. Synthetic DNA linkers were ligated to the 5' and 3' ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E. coli trp promoter. Transformation of E. coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1. Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted Mr. Unglycosylated PAI-1 from E. coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37 degrees C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator. Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography. The purified latent PAI-1 was crystallized.

Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli.

A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli. mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx. 30% of total cellular protein produced by the recombinant strain. A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water. This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract. The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2. No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha. The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay. In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms). The truncated species were isolated and found to have the same biological activity as the complete polypeptide. Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.

The isolation and characterization of the pyruvate kinase-encoding gene from the yeast Yarrowia lipolytica.

The dimorphic yeast, Yarrowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (1) the nucleotide sequence of the proposed PYK gene predicts a protein that is highly homologous to the corresponding Saccharomyces cerevisiae enzyme, and (2) PYK-specific activity was increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. lipolytica gene.

Characterization of a plasmid determining resistance to erythromycin, lincomycin, and vernamycin Balpha in a strain Streptococcus pyogenes.

A plasmid determining resistance to erythromycin, lincomycin, and vernamycin B(alpha) was isolated from a strain of Streptococcus pyogenes. The plasmid has a molecular weight of approximately 17 x 10(6) and is present to the extent of one to two copies per chromosomal genome equivalent.

Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid.

Streptococcus faecalis strain DS16 harbors the conjugative hemolysin-bacteriocin plasmid pAD1 (35 megadaltons) and the nonconjugative R-plasmid pAD2 determining resistance to streptomycin, kanamycin, and erythromycin; a tetracycline resistance (Tetr) determinant is located on the chromosome. When strain DS16 was mated (on membrane filters) with the plasmid-free strain JH2-2, Tetr transconjugants could be obtained at a frequency of about 10(-6) per recipient. Analyses of transconjugants showed that some contained the Tetr determinant linked to pAD1. Subsequent studies showed that the Tetr determinant was located on a 10-megaldalton transposon, designated Tn916, which could insert into two hemolysin plasmids: pAM gamma 1 and pOB1. In addition, derivatives of DS16 devoid of pAD1 were capable of transferring Tetr to recipient strains. Transconjugants (plasmid-free) from such matings could subsequently act as donors in the transfer of Tetr. Both transposition and transfer were found to be rec independent.

Plasmid-determined resistance to erythromycin: comparison of strains of streptococcus faecalis and streptococcus pyogenes with regard to plasmid hmology and resistance inducibility.

Streptococcus faecalis strains DS-5 and Streptococcus pyogenes strain AC-1 both have a 17 million dalton plasmid that determines resistance to erythromycin, lincomycin, and vernamycin B(alpha). The results of deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments indicate that the two plasmids are about 95% homologous. It was also shown that erythromycin resistance is inducible in AC-1 and constitutive in DS-5.

Mutational and immunochemical analysis of plasminogen activator inhibitor 1.

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.

DNA sequence of a major human leukocyte interferon gene.

The gene for human leukocyte interferon alpha 2 (designated either LeIF A or HuIFN-alpha 2) has been isolated from a human genome library. The DNA sequence of this gene demonstrates that it lacks introns. The 3' noncoding sequences of the IFN-alpha 2 gene correspond to two types of IFN-alpha 2 cDNA clones we have isolated that have alternate sites of polyadenylylation. A comparison of seven human IFN-alpha sequences shows that they are homologous in the 5' flanking region and contain identical "TATA box" sequences. The recombinant lambda clone containing the IFN-alpha 2 gene also contains two copies of the "Alu family" repeat sequence.

Human fibroblast interferon gene lacks introns.

A recombinant lambda bacteriophage isolated from a human genome library contains the gene for fibroblast interferon (IFN-beta1). The DNA sequence of this gene is identical to the sequence of its mRNA and is devoid of introns.

Carboxyterminal region of hybrid leukocyte interferons affects antiviral specificity.

Four hybrid human leukocyte interferon (IFN-alpha) genes have been constructed and expressed in Escherichia coli using molecular cloning methods. Plasmids containing genes encoding human interferons, IFN-alpha A, IFN-alpha D, IFN-alpha I and several hybrids of the different IFN-alpha genes (formed by in vitro recombination at common restriction endonuclease sites located within the DNA sequence encoding mature polypeptides) joined identically to an E. coli trp promoter gave rise to bacterial-produced interferons with distinctly different antiviral activities. The expression plasmid which directs the synthesis of IFN-alpha I was constructed using a gene isolated from a human genomic library in a manner similar to the previous expression of IFN-alpha A and IFN-alpha D cDNA clones. The use of a cell-free transcription-translation system has allowed the calculation of the specific activities of IFN-alpha made from isolated DNA fragments containing these hybrid IFN-alpha genes. These bacterially-derived interferons vary considerably in their ability to inhibit vesicular stomatitis virus (VSV) in different mammalian cells. The results show that the cloned hybrid interferons have unique antiviral activities when compared with the parent interferons, and they demonstrate that more active IFN-alpha s can be made using recombinant DNA techniques.

Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917.

Plasmids pAD1 (37.8 megadaltons) and pAD2 (17.1 megadaltons) of Streptococcus faecalis strain DS16 have been mapped with restriction enzymes. The location of a hemolysin-bacteriocin determinant on the conjugative pAD1 plasmid was derived from analyses of transposon insertions. Electron microscope and hybridization analyses located Tn917(Em) and the streptomycin (Sm) and kanamycin (Km) resistance determinants on the nonconjugative pAD2 plasmid. It was shown previously that the erythromycin (Em) resistance associated with Tn917 is inducible and that transposition from pAD2 to pAD1 is also stimulated by exposure of cells to low concentrations of Em. Here we show that inducing concentrations of Em also increase the conjugative transfer potential of pAD1; this is possibly related to a mild and short-lived inhibitory stress placed on the cells before full induction of resistance. Selection of Em-resistant transconjugants arising from matings between DS16 and a plasmid-free recipient gave rise to transconjugants which primarily harbor stable pAD1::pAD2 cointegrates. A 30-min exposure of donors to Em (0.5 microgram/ml) before mating resulted in a severalfold increase in the number of such transconjugants. However, a small fraction (e.g., 3 of 40) of these Emr Smr Kmr transconjugants harbored pAD1::Tn917 and pAD2 molecules. Since we believe pAD2 is incapable of being mobilized by pAD1 without being covalently linked, it is likely that transfer in these cases involved cointegrates representing structural intermediates in the transposition of Tn917 from pAD2 to pAD1. It follows that such intermediates probably had two copies of Tn917 and readily resolved after transfer. (These cointegrates are different from the stable cointegrates which were shown to have only a single copy of Tn917; the latter are assumed not to be related to transposition.) Two variants with altered Tn917 transposition properties were derived. One of them transposed at an elevated frequency, whereas the other showed no detectabel transposition. In neither case was transposition influenced by Em exposure; however, both remained inducible for Em resistance.

The sequence of human serum albumin cDNA and its expression in E. coli.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

The Yarrowia lipolytica LEU2 gene.

A 2810 bp DNA fragment containing the beta-isopropylmalate dehydrogenase gene of the dimorphic yeast Yarrowia lipolytica has been sequenced. The sequence contains an open reading frame of 405 codons, predicting a protein of 43,366 molecular weight. Protein sequence homology with the polypeptide encoded by the LEU2 gene of Saccharomyces cerevisiae is 64%, whereas DNA sequence homology is 61%. The 5'- and 3'-flanking regions of the Y. lipolytica LEU2 gene share only some general structural features common to genes of S. cerevisiae such as the presence and location of TATA boxes, CAAT boxes, CACACA repeats, the lack of G residues in the 5'-untranslated region and 3'-transcription terminators. Transcription of a 1.4 kb mRNA begins at a small cluster of sites approximately 40 base pairs before the initial ATG.

In vivo augmentation of IFN-gamma with a rIL-12 human/mouse chimera: pleiotropic effects against infectious agents in mice and rats.

A heterodimer containing the mouse 35 kDa and human 40 kDa subunit of IL-12 was expressed in COS cells (cIL-12). Administration of 25-200 U of the cIL-12-COS supernatant to mice twice daily for 2 days augmented spleen cell IFN-gamma production in response to IL-2 and peritoneal macrophage activity (superoxide and nitrites) as compared to animals receiving mock transfected supernatants. cIL-12 also increased levels of IFN-gamma in serum but most dramatically following an LPS injection (50-fold over controls). Animals pretreated with cIL-12 suffered enhanced mortality following challenge with the Gram negative organism E. coli but enhanced survival or clearance following infection with the Gram positive organisms L. monocytogenes and S. aureus. Although daily treatment of mice with cIL-12 following an intranasal influenza A infection elevated levels of IFN-gamma in the bronchioalveolar lavage fluid three-fold over controls, neither prophylactic or therapeutic treatment with the same dose level decreased viral titres in the lung. In addition, no effect was observed in animals infected with encephalomyocarditis virus or respiratory syncytial virus. Therefore, cIL-12 is a potent in vivo augmentor of IFN-gamma production. It has differential effects on infectious disease depending on the invading organism and time of administration; being efficacious for intracellular bacteria but ineffective at the same dose levels against viral diseases.

Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica.

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidase-processing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.