Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter

To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300 000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lacUV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.     

Expression of human alpha-fetoprotein in yeast.

Human alpha-fetoprotein (AFP) was expressed in Saccharomyces cerevisiae, with a plasmid containing the cDNA sequence for human AFP fused with the rat AFP signal peptide. The recombinant AFP was purified from the yeast lysate by DEAE-cellulose and immunoaffinity chromatography. The amino acid composition and the molecular weight of the recombinant AFP were similar to those of hepatoma AFP. N-terminal amino acids sequence analysis indicated that the signal peptide had been processed. The recombinant and hepatoma AFP reacted identically in Ouchterlony immunodiffusion and radioimmunoassay tests. These observations indicated that the yeast recombinant protein had the properties of native AFP.     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

Unusual zymogen-processing properties of a mutated form of prochymosin

Site-specific mutagenesis of the gene encoding bovine prochymosin was used to produce a mutated zymogen in which seven contiguous amino acids of the N-terminal propeptide had been deleted and an eighth residue had been substituted. This altered region spans the normal site of autocatalytic proteolysis that occurs at the same time as (enzymatic) activation of prochymosin at acidic pH. Activation of the mutated zymogen at pH 4.5 was extremely slow, and cleavage occurred at an unusual Ser-Lys bond in the propeptide of the zymogen. The mutated prochymosin incubated at pH 2 generated the usual pseudochymosin by cleavage of the normal Phe-Leu bond, but at a rate severalfold slower than the authentic zymogen. These results indicate that even after deletion of seven of 42 amino acids of the propeptide the mutant protein could still assume a prochymosin (zymogen) structure, although these changes did result in striking differences in acid-catalyzed activation and processing reactions at one but not the other of the two processing sites of prochymosin.     

Norwalk virus N-terminal nonstructural protein is associated with disassembly of the Golgi complex in transfected cells

Norwalk virus is the prototype strain for members of the genus Norovirus in the family Caliciviridae, which are associated with epidemic gastroenteritis in humans. The nonstructural protein encoded in the N-terminal region of the first open reading frame (ORF1) of the Norwalk virus genome is analogous in gene order to proteins 2A and 2B of the picornaviruses; the latter is known for its membrane-associated activities. Confocal microscopy imaging of cells transfected with a vector plasmid that provided expression of the entire Norwalk virus N-terminal protein (amino acids 1 to 398 of the ORF1 polyprotein) showed colocalization of this protein with cellular proteins of the Golgi apparatus. Furthermore, this colocalization was characteristically associated with a visible disassembly of the Golgi complex into discrete aggregates. Deletion of a predicted hydrophobic region (amino acids 360 to 379) in a potential 2B-like (2BL) region (amino acids 301 to 398) near the C terminus of the Norwalk virus N-terminal protein reduced Golgi colocalization and disassembly. Confocal imaging was conducted to examine the expression characteristics of fusion proteins in which the 2BL region from the N-terminal protein of Norwalk virus (a genogroup I norovirus) or MD145 (a genogroup II norovirus) was fused to the C terminus of enhanced green fluorescent protein. Expression of each fusion protein in cells showed evidence for its colocalization with the Golgi apparatus. These data indicate that the N-terminal protein of Norwalk virus interacts with the Golgi apparatus and may play a 2BL role in the induction of intracellular membrane rearrangements associated with positive-strand RNA virus replication in cells.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

Expression and properties of prochymosin derivatives containing extensions of various length in the pro-part

A series of hybrid prochymosin derivatives containing portions of the simian virus 40 small-t antigen in the pro-part was constructed. Portions comprising 93, 63, 47, 12, and 1 amino acid (aa) from the N terminus of the small-t antigen were separately fused via eight polylinker-encoded amino acids to a prochymosin product commencing with the 5th aa of the pro-part. All the DNAs coding for the hybrid proteins were put under pL-promoter control in the expression constructs. Expression revealed that only fusion of the 47-aa or 12-aa stretch of the small-t antigen to prochymosin gave stable protein products and that only the latter one allowed the hybrid prochymosin to be activated to chymosin. The products containing 93 aa and 63 aa of small-t antigen were unstable and degraded. Complete removal of the small-t antigen portion led to mRNA instability, probably owing to inefficient initiation of translation.     

cDNA clones completing the nucleotide and derived amino acid sequence of the alpha 1 chain of basement membrane (type IV) collagen from mouse

Six cDNA clones add 3549 nucleotides to the DNA sequence of the alpha 1 chain of basement membrane (type IV) collagen. Thus the complete nucleotide and derived amino acid sequence of the alpha 1 type IV collagen with 5007 nucleotides coding for 1669 amino acids with a calculated Mr of 160,827 is known. The six cDNA clones cover the putative N-terminal signal peptide, the 7 S region and two thirds of the helical region extending into the previously published murine nucleotide sequence [(1986) Gene 43, 301]. The protein sequence for 289 amino acids of the helical region adjacent to the 7 S region has not previously been published for any species.     

Molecular cloning and expression in yeast of caprine prochymosin

We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.     

Molecular cloning of a splicing variant of human RECQL helicase

A cDNA was isolated from the human heart library. This cDNA variant was produced by the deletion of 176 bases at the 5(') end of human RecQL gene, presumably by an alternative mRNA splicing. The cDNA contains two open reading frames and so may encode two isoforms of human RECQL. The first isoform is a 105 amino acid protein with the first 53 N-terminal amino acids identical to the known sequence of RECQL protein and followed by 52 amino acids introduced by in-frame premature stop codon. The second isoform is a 537 amino acid protein that has the same sequence as the published human RECQL helicase, except the first 112 amino acids at the N-terminal end were absent. The possible roles of both of these proteins are discussed.     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.     

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.     

Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72.

A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.     

cDNA cloning and structure of mouse putative Ah receptor.

Mouse cDNA clones for a putative Ah receptor have been isolated from a cDNA library of mRNA from Hepa-1 cells by an oligonucleotide probe produced by PCR with a pair of primers which was synthesized according to the reported N-terminal sequence of 26 amino acids. The cDNA clones encode a polypeptide of 805 amino acids with a helix-loop-helix motif and with some similarity to a certain region designated PAS of Drosophila Per and Sim, and human Arnt protein. Cotransfection of an expression vector of the Ah receptor with a reporter plasmid pMC6.3k consisting of CYP1A1 promoter and CAT structural gene into CV-1 cells enhanced the CAT expression in response to added 3-methylcholanthrene.     

Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.