Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor fromSorghum bicolor seedlings

A 711-bp cDNA encoding a cysteine proteinase inhibitor (cystatin) was isolated from a cDNA library prepared from 7–10 cmSorghum bicolor seedlings. The nearly full-length cDNA clone encodes 130 amino acid residues, which include the Gln-Val-Val-Ala-Gly motif, conserved among most of the known cystatins as a probable binding site for cysteine proteinases. The amino acid sequence of sorghum cystatin deduced from the cDNA clone shows significantly homology to those of other plant cystatins. The sorghum cystatin expressed inE. coli showed a strong papain-inhibitory activity.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds

Sunflower cystatin a (Sca) is distinguished from other phytocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily conserved Gly residue. The cDNA encoding Sca was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with the Sca protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sca is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sca. To address this assumption and also to investigate the significance of the N-terminal extension sequence to Sca for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sca, and a recombinant matured Sca (rSca) were overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE that was the same as rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase

The cDNA encoding human ferrochelatase [EC 4.99.1.1] was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA. The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr. 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails. Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr. 42,158) with a putative leader sequence of 54 amino acid residues. The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme. Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells. As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.     

Molecular cloning and sequence analysis of a cDNA encoding a porcine kidney renin-binding protein

A complementary DNA encoding a renin-binding protein (RnBP) has been isolated from a porcine kidney cDNA library by immunological screening of in vitro translation products from the cDNAs. Analysis of the nucleotide sequence of the clone revealed a 1,342-nucleotide sequence with a 5'-terminal untranslated region of 52 nucleotides, an open reading frame of 1,206 nucleotides that encodes 402 amino acids, and a 3'-terminal untranslated region of 84 nucleotides that contains the polyadenylation signal sequence, AATAAA. The predicted amino acid sequence contains no hydrophobic amino-terminal sequence and does not show significant homology to those of other identified proteins. The in vitro translated RnBP was found to have the same molecular weight, 42,000, as that of the purified RnBP from porcine kidney on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and it formed a complex with renin purified from porcine kidney, which indicates that the cDNA encodes a functional RnBP without a propeptide sequence. The RnBP cDNA probe hybridized to a 1.5-kilobase mRNA in kidney, liver, adrenal, and pituitary glands, the amount being much greater in kidney than in the other tissues. Southern blot analysis showed the presence of a unique gene for RnBP in the porcine genome.     

Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase

A complete cDNA clone encoding human cholesterol 7 alpha-hydroxylase has been isolated using a rat P-450ch7 alpha cDNA insert [(1989) FEBS Lett. 257, 97-100] as a probe and totally sequenced. The cDNA contained 1512-base pair open reading frame encoding 504 amino acid residues (Mr 57,630), 39-base pair 5'-untranslated region 1322-base pair 3'-ultranslated region including 20 nucleotides of poly A tail in the total length of 2873 base pairs. The deduced amino acid sequence showed 82% similarity to rat P-450ch7 alpha. Unique amino acid residues were observed in putative binding domains for heme and steroid which are highly conserved in most steroidogenic P-450s.     

Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase

lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.     

Molecular cloning and sequence analysis of cDNA encoding human kidney D-amino acid oxidase

cDNA clones encoding D-amino acid oxidase were isolated from a human kidney cDNA library by hybridization with cDNA for the pig enzyme. The cDNA insert of 2.0 kilobase pairs long provided coding information for a protein consisting of 347 amino acids. The molecular mass of the enzyme was calculated to be 39,410 Da. The amino acid sequence similarity between the pig and human enzymes is 84.4%, and among the active site residues proposed from chemical modification studies, methionine-110 of the pig enzyme was replaced by threonine. Northern blot analysis confirmed the expression of an mRNA of 2.0 kilobases encoding the D-amino acid oxidase in human kidney.     

三疣梭子蟹蜕皮抑制激素cDNA的克隆与序列分析

甲壳动物的蜕皮是由位于头胸部前鳃腔的一对Y-器通过分泌蜕皮激素（Molting hormone）来控制的（Lachaise et al．,1993）,而蜕皮激素的分泌又受到蜕皮抑制激素（Molt-inhibiting hormone,MIH）的调控（Watson et al．,2001）。MIH和性腺抑制激素（Gonad-inhibiting hormone,GIH）、甲壳动物高血糖激素（Crustacean hyperglycemic hormone,CHH）、     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.     

Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists

The beta-adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catecholamines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of untreated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when purified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first example of cysteine proteinase inhibitors that can be induced by beta-adrenergic agonists.     

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.     

Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790

A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.     

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor,cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.