Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin

Harris, T. J. R.; Lowe, Peter A.; Lyons, A. Bruce; Thomas, Philip; Eaton, Michael A. W.; Millican, T. A.; Patel, T.P.; Bose, Chhanda; Carey, N. H.; Doel, M.T.

doi:10.1093/nar/10.7.2177

Cited by 102 publications

(22 citation statements)

References 26 publications

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“…3). This was much larger than expected for a precursor protein of Mr 53,000 (27) and also much larger than the 1.5-to 1.6-kb message lengths for renin, pepsinogen (31), and chymosin (32). The larger size of the cathepsin D message has been shown here to be due to a long 3' untranslated region.…”

Section: Isolation Of a Partial Cdna Clone For Human Cathepsin Dmentioning

confidence: 46%

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Cloning and sequence analysis of cDNA for human cathepsin D.

Faust¹,

Kornfeld²,

Chirgwin³

1985

Proc. Natl. Acad. Sci. U.S.A.

283

138

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An 1110-base-pair cDNA clone for human cathepsin D was obtained by screening a XgtlO human hepatoma G2 cDNA library with a human renin exon 3 genomic fragment. Poly(A)+ RNA blot analysis with this cathepsin D clone demonstrated a message length of about 2.2 kilobases. The partial clone was used to screen a size-selected human kidney cDNA library, from which two cathepsin D recombinant plasmids with inserts of about 2200 and 2150 base pairs were obtained. The nucleotide sequences of these clones and of the XgtlO clone were determined. The amino acid sequence predicted from the cDNA sequence shows that human cathepsin D consists of 412 amino acids with 20 and 44 amino acids in a pre-and a prosegment, respectively. The mature protein region shows 87% amino acid identity with porcine cathepsin D but differs in having nine additional amino acids. Two of these are at the COOH terminus; the other seven are positioned between the previously determined junction for the light and heavy chains of porcine cathepsin D. A high degree of sequence homology was observed between human cathepsin D and other aspartyl proteases, suggesting a conservation of three-dimensional structure in this family of proteins.Cathepsin D is a lysosomal endoprotease that is present in all mammalian cells (for review, see ref. 1). It is a member of the aspartyl protease family, among which are the well-studied secretory enzymes renin, pepsin, and chymosin (2). Cathepsin D is the only aspartyl protease known to be lysosomal rather than secretory. Recently, the complete amino acid sequence for the mature protein of porcine cathepsin D has been determined (3). Alignment of this sequence with that of renin showed an overall 48.9o homology (3). In addition, the region surrounding the first activesite aspartyl (residue 32 in the pepsin numbering convention) was even more highly conserved.The entire human renin gene has now been cloned and its organization has been determined (4, 5 A human kidney cDNA library was screened for a fulllength clone. Total RNA was isolated by the guanidinium thiocyanate method (7) from a portion of surgically removed, perfused kidney and twice selected by chromatography on an oligo(dT)-cellulose column (8). Poly(A)+ RNA (29 ,ug) was reverse-transcribed under the conditions of Retzell et al. (9). The first-strand heteroduplex was converted to fully doublestranded cDNA by replacement synthesis (10). The doublestranded cDNA was then resolved by preparative electrophoresis in an agarose gel and cDNAs 1.8-2.5 kilobases (kb) long were cut out and isolated (11). These cDNAs were then oligo(dC)-tailed (12) and annealed with plasmid pUC19 (13) that had been oligo(dG)-tailed at the Sac I site. E. coli K-12 strain DH-1 (14) was transformed and filter replicas of ampicillin-resistant colonies were prepared by the method of Hanahan and Meselson (15 Filters were washed at 45°C in 0.1 x NaCl/Cit/0.1% NaDodSO4 prior to autoradiography. Positive phage were plaque-purified (11) at least three times.Abbreviations: kb, kilobase(s); bp, bas...

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Section: Isolation Of a Partial Cdna Clone For Human Cathepsin Dmentioning

confidence: 46%

“…Alignment of this sequence with that of renin showed an overall 48.9o homology (3). In addition, the region surrounding the first activesite aspartyl (residue 32 in the pepsin numbering convention) was even more highly conserved.…”

mentioning

confidence: 92%

Cloning and sequence analysis of cDNA for human cathepsin D.

Faust¹,

Kornfeld²,

Chirgwin³

1985

Proc. Natl. Acad. Sci. U.S.A.

283

138

View full text Add to dashboard Cite

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“…Calf prochymosin, the zymogen of chymosin, a milk-clotting aspartic proteinase used in cheese production, has been studied here as a model of such proteins. Prochymosin cDNA has been cloned and expressed in Escherichia coli (Harris et al, 1982;Emtage et al, 1983); like many other animal proteins expressed in E. coli, recombinant prochymosin forms intracellular protein aggregates, referred to as inclusion bodies or refractile granules (Emtage et al, 1983;Nishimori et al, 1984;Schoemaker et al, 1985). Formation of such aggregates can simplify the early stages of concentration and purification of the recombinant products, but, in general, problems are encountered in the recovery of biologically active proteins from them in acceptable yield.…”

Section: Introductionmentioning

confidence: 99%

Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Sugrue

Marston²,

Lowe³

et al. 1990

Biochemical Journal

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1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v. c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.

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“…Several aspartic protease genes from molds and bacteria have been cloned and expressed (15,22,33,44). Bovine chymosin has been cloned and successfully expressed in Escherichia coli (11,18,25,27), in Bacillus spp. (19,28), in yeasts (14,24,38,42), and in filamentous fungi (7,40,41).…”

mentioning

confidence: 99%

Cloning and Expression of clt Genes Encoding Milk-Clotting Proteases from Myxococcus xanthus 422

Poza

Prieto-Alcedo

Sieiro

et al. 2004

Appl Environ Microbiol

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The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.Currently, there are three main types of rennins used by the cheese-making industry: (i) rennins extracted from the abomasum of suckling ruminants (such as chymosin [EC 3.4.23.4]), (ii) rennins prepared from microbial broths, and (iii) recombinant rennins. In general, animal rennins are not sufficient to cover world demands, and this fact has prompted research into both microbial and recombinant rennins. Although a variety of bacteria, yeasts, and fungi have been isolated as natural producers of milk-coagulating enzymes (3,10,13,26,32,45), only two genera are used worldwide in cheese production: Mucor (12, 39) and Endothia (8, 15). Several aspartic protease genes from molds and bacteria have been cloned and expressed (15,22,33,44). Bovine chymosin has been cloned and successfully expressed in

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Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin

Cited by 102 publications

References 26 publications

Cloning and sequence analysis of cDNA for human cathepsin D.

Cloning and sequence analysis of cDNA for human cathepsin D.

Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Cloning and Expression of clt Genes Encoding Milk-Clotting Proteases from Myxococcus xanthus 422

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