A cDNA library prepared from human liver was screened for alpha 1-antitrypsin, a major constituent of plasma which functions as inhibitor of proteolytic enzymes. The library was screened using a 12-base-long synthetic oligodeoxyribonucleotide corresponding to a known DNA fragment of human alpha 1-antitrypsin and by hybrid-selection of alpha 1-antitrypsin mRNA. A plasmid, pULB1523, was identified carrying a cDNA insert of about 1400 bp coding for human alpha 1-antitrypsin. Restriction mapping and DNA sequence analysis indicated that the 1400 bp code for the signal peptide and for the complete mature alpha 1-antitrypsin molecule. In addition, a solid-phase enzyme-linked immunoassay showed that pULB1523 expresses human alpha 1-antitrypsin in bacteria. Fusion of the alpha 1-antitrypsin sequence to the leader sequence of the beta-lactamase gene (plasmid pKT287) resulted also in the expression of the protein in bacteria.
Nucleotide sequences coding either for the precursor or the mature form of human a1-antitrypsin have been placed under the control of the yeast ARG3 expression signals. Recombinant plasmids pRIT10782 and pRIT10787 express the precursor or the mature a1-antitrypsin species, respectively, in two different yeast strains, with yields ranging between 0.3 and 1% of total soluble proteins. The a1-antitrypsin synthesized in yeasts was specifically recognized by polyclonal and monoclonal antibodies raised against human al-antitrypsin. In addition, it was shown to be biologically active in its mature form only, with optimal activity in a peptidase-deficient yeast strain. Plasmids used in this work, pULB1523, YEp13, pMC200, and pRIT10749, have been described (9,(12)(13)(14). Yeast strains Ic1697d (argJ+, leu2) and 1OS44c (leu2-3, 1eu2-112, pep4-3) were grown in yeast nitrogen base medium (Difco) supplemented, when needed, with 4 ,ug of arginine per ml. Transformations of yeast strains and of E. coli strain MM294 (endoA, thi-, hsdR, supE) were done as described (15, 11). Colony hybridization experiments follow the procedure of Grunstein and Hogness (16).Plasmid DNA purification was achieved on cesium chloride gradients and small-scale DNA preparations for rapid analysis were performed as described (11). DNA fragments were analyzed and purified either on agarose or polyacrylamide gels (11).Preparation of Yeast Extracts. Yeast extracts for the immunoassay and for the activity assay of a1-antitrypsin were prepared as follows: 200-nil cultures of appropriate strains were cultivated at 30°C up to OD620 of about 0.4. Cells were collected by centrifugation, washed in distilled water, resuspended in 10 ml of extraction buffer (50 mM Tris HCl, pH 8/1 mM EDTA/0.1 mM cysteine), and disrupted by two passages through a French pressure cell at 12,000 psi (1 psi = 6895 Pa). Extracts were then centrifuged for 30 min at 15,000 rpm (Sorvall SS.34) to eliminate cell debris, and supernatants were assayed for protein concentration by using the procedure of Lowry et al. (17). Supernatants were immediately assayed or frozen at -20°C until used.Expression Assay for al-Antitrypsin. Immunological detection and quantification of al-antitrypsin were performed by micro-ELISA using either polyclonal or monoclonal antibodies against a1-antitrypsin as described (9,10).Assay for al-Antitrypsin Activity. The assay measures the inhibitory capacity of al-antitrypsin toward trypsin. It consists of a microtest using the chromogenic substrate S2444 (L-pyroglutamylglycyl-L-arginine, p-nitroanilide hydrochloride, Kabi Diagnostica, Stockholm, Sweden). The polystyrene microtest plates were incubated for 1 hr with 1% bovine serum albumin and washed extensively with distilled water. al-Antitrypsin at various concentrations was incubated with a fixed amount of trypsin for 20 min at 37°C in a 200-,ul final reaction volume of (0.1 M Tris HCl, pH 8.2/0.15 M NaCl/0.01 M EDTA/0.5% polyethylene glycol, Mr 6000). Samples were cooled slowly at room temperature and then...
A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.
Complementary DNA coding for human α1‐antitrypsin has been placed under the control of the λPR promotor carrier by the expression vector pCQV2 [1]. In conditions which allow transcription from this promotor (thermoinactivation of the repressor), Escherichia coli cells harbouring the recombinant plasmid pULB1114 express human α1‐antitrypsin (± 9000 molecules/cell). The product has a M r of 44 000, corresponding to mature unglycosylated α1‐antitrypsin.
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