Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization.

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli.

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.

Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clones.

Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.

Expression of cloned human haptoglobin and alpha 1-antitrypsin complementary DNAs in Saccharomyces cerevisiae.

Nucleotide sequences coding either for human preprohaptoglobin or for prohaptoglobin have been placed under the control of yeast ARG3 expression signals. Recombinant plasmids pRIT12598 and pRIT12597 express the prepro- and the pro-form of alpha 2 beta haptoglobin respectively, but at very low levels. Comparison with the expression of human pre- and mature alpha 1-antitrypsin cDNAs, cloned in the same expression vector, reveals large differences in the levels of specific proteins produced in yeast, although specific mRNA levels are similar. It is shown that presence or absence of the signal sequence in the cDNA construction results in a 20- to 30-fold difference in the yields of heterologous products. However, since haptoglobin and alpha 1-antitrypsin behave differently, the difference in expression for prohaptoglobin compared with the expression of mature alpha 1-antitrypsin is about three orders of magnitude. In addition, we provide evidence that glycosylation of both proteins can occur in yeast only when the signal sequence is present in the DNA constructions.

The alpha 2 cDNA sequence of human haptoglobin carries a bacterial promoter functional in vivo.

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete alpha 2FS beta precursor protein or only for the beta subunit have been placed under the control of the lambda PR promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the PR promoter, the complete alpha 2FS beta constructions constitutively express a smaller polypeptide of approximately 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (alpha 2 PF and alpha 2 PS) located in the duplicated alpha 2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp alpha 2 cDNA sequence, when fused upstream to the cDNA coding for alpha 1-antitrypsin, constitutively promotes in vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against alpha 1-antitrypsin or haptoglobin.

Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNA.

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.

Expression of human alpha 1-antitrypsin cDNA in the yeast Saccharomyces cerevisiae.

Nucleotide sequences coding either for the precursor or the mature form of human alpha 1-antitrypsin have been placed under the control of the yeast ARG3 expression signals. Recombinant plasmids pRIT10782 and pRIT10787 express the precursor or the mature alpha 1-antitrypsin species, respectively, in two different yeast strains, with yields ranging between 0.3 and 1% of total soluble proteins. The alpha 1-antitrypsin synthesized in yeasts was specifically recognized by polyclonal and monoclonal antibodies raised against human alpha 1-antitrypsin. In addition, it was shown to be biologically active in its mature form only, with optimal activity in a peptidase-deficient yeast strain.

Expression of human alpha 1-antitrypsin in Escherichia coli.

Complementary DNA coding for human alpha 1-antitrypsin has been placed under the control of the lambda PR 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 alpha 1-antitrypsin (+/- 9000 molecules/cell). The product has a Mr of 44000, corresponding to mature unglycosylated alpha 1-antitrypsin.

Cloning and expression in Escherichia coli of full-length complementary DNA coding for human alpha 1-antitrypsin.

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.