Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.     

cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle

A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids. The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases. The recombinant prolyl oligopeptidase generated in E. coli was purified and its properties were examined. The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme. Northern blot analysis showed wide expression of the prolyl oligopeptidase gene. Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase. These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression.     

A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin: cloning, expression, and identification of catalytic serine residue

The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (K(i) = 0.7 0.14 microM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.     

Isolation and sequencing of a genomic clone encoding aspartic proteinase of Rhizopus niveus.

A gene encoding Rhizopus niveus aspartic proteinase was isolated from an R. niveus genomic library by using oligonucleotides probes corresponding to its partial amino acid sequence, and its nucleotide sequence was determined. By comparing its deduced amino acid sequence with the amino acid sequence of rhizopuspepsin (5, 26), we concluded that the R. niveus aspartic proteinase gene has an intron within its coding region and that it has a preproenzyme sequence of 66 amino acids upstream of the mature enzyme of 323 amino acids.     

Cloning and mapping of a testis-specific gene with sequence similarity to a sperm-coating glycoprotein gene

A testis-specific gene Tpx-1, located between Pgk-2 and Mep-1 on mouse chromosome 17, was isolated from a cosmid clone, and its cDNA sequences were determined. The predicted coding sequence of Tpx-1 isolated from BALB/c mice showed 64.2% nucleotide and 55.1% amino acid sequence similarity with that of a rat sperm-coating glycoprotein gene, the protein product of which is secreted by the epididymis. To examine the evolutionary relationship between Tpx-1 and a sperm-coating glycoprotein gene, the cDNA sequence of TPX1, the human counterpart of Tpx-1, was determined. The comparison of the predicted coding sequences of Tpx-1 and TPX1 showed 77.8% nucleotide and 70% amino acid sequence similarity. Since Tpx-1 (from mouse) is more similar to TPX1 (from man) than it is to a rat sperm-coating glycoprotein gene, we conclude that Tpx-1 (TPX1) and a sperm-coating glycoprotein gene are closely related, but distinct, genes belonging to the same gene family. The predicted Tpx-1 protein of a t mutant mouse CRO437 differs from that of BALB/c mice by one amino acid insertion in the putative signal peptide. TPX1 was mapped to 6p21-qter by Southern blot analysis of interspecies somatic hybrid cell lines.     

Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase

We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactis Wg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.     

人促红细胞生成素受体胞外区基因的克隆及其在大肠肝菌中的表达

以人胎肝为材料,通过ＰＴ－ＰＣＲ的方法扩增出人促红细胞生成素受体（ｈＰｅｏＲ）的胞外区基因。将获得的或熟体胞外区基因起始密码子改构后克隆原核表达载体ｐＢＶ２２０中,进行原核温控诱导表达。表达产物经蛋白Ｎ端测序及Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验证实表达产物是ｈＥｐｏＲ胞外区蛋白。利用上罐发酵培养获得的包涵体蛋白经复性纯化后,体外生物学活性检测表明表达产物可特 抑制ＴＦ－１细胞在Ｅｐｏ刺激下的生长,证实了     

Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.     

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.     

Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx mori

ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis.     

Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice Proteinase Inhibitor

With the primers designed basing on the terminal amino acid sequences of rice proteinase inhibitors and the preferred codons of rice genes, a new gene coding for a rice proteinase inhibitor has been amplified and cloned from Oryza sativa var. japonica (cv. Zhonghua 8) using PCR technique. The gene contains 408 basepairs and encodes 133 amino acid residues. The deduced amino acid sequence with duplicated Bowman-Birk type structure and active sites specific to trypsin has relatively high homology with that of proteinase inhibitors from wheats, beans etc. As for rice, the new gene shares 74.8% homology with a rice bran trypsin inhibitor reported previously. The evolutionary characteristics of the proteinase inhibitor family has also been discussed.     

一种水稻蛋白酶抑制剂基因的克隆及其结构分析

参照水稻蛋白酶抑制剂部分氨基酸序列 ,利用水稻偏爱密码子设计引物 ,经 PCR扩增 ,从我国水稻 (Oryza sativa)品种“中花 8号”中克隆到一个长 40 8bp的基因。序列测定和分析表明 ,克隆到的是一个未见报道的新的水稻蛋白酶抑制剂基因 ,该基因编码了一个由 1 33个氨基酸组成 ,具有重复双功能结构域和以抑制胰蛋白酶为主的活性中心的包曼 -伯克 (Bowman- Birk)型蛋白酶抑制剂 ,该基因推导的氨基酸序列与大麦、小麦、豆类等的某些蛋白酶抑制剂的氨基酸序列具有较高的同源性 ,与该家族的水稻的一种胰蛋白酶抑制剂氨基酸全序列同源性高达 75%。     

Drosophila CTLA-2-like protein (D/CTLA-2) inhibits cysteine proteinase 1 (CP1), a cathepsin L-like enzyme

In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine proteinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.     

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.     

一个富含谷氨酸的人类分泌蛋白基因hMGRAP的克隆与表达分析

为了筛选有功能意义的新的分泌蛋白,并对其功能进行探索,采用生物信息学工具预测得到一个新的人类分泌蛋白基因hMGRAP(Human Multiple Glutamine Repeat Acidic Protein)。该基因定位于染色体7q22.1,全长cDNA为1547bp,编码含有248个氨基酸的蛋白,该蛋白富含重复的谷氨酸序列, 等电点为4.6。用PCR方法从正常人的混合cDNA文库中克隆到hMGRAP。 Western blot实验表明hMGRAP能大量地从瞬时转染的cos-7细胞中分泌到细胞培养液中。RT-PCR结果显示,hMGRAP相对表达较高的组织为睾丸、骨骼肌和肾。总之,筛选并克隆到一个新的人类分泌蛋白基因hMGRAP,其生物学功能可能因其重复的谷氨酸编码序列而具有一定特殊性。Abstract: To search for human novel secreted proteins and study their biological functions, using bioinformatical tools and experimental approaches, a novel secreted protein, human hMGRAP (Human Multiple Glutamine Repeat Acidic Protein) was obtained. hMGRAP consists of six coding exons spanning 1547bp of genomic DNA on the human chromosome 7q22.1, which encodes a protein with 248 amino acids. hMGRAP is rich of glutamic acid repeated sequence and the PI is 4.6. The coding sequence of hMGRAP was cloned by PCR method from the cDNA pool composed of nine human tissues. Western blot showed that hMGRAP protein was massively secreted out from the transiently transfected Cos-7 cells. RT-PCR result indicated hMGRAP mRNA was abundantly expressed in testis. In summary, a novel human gene encoding a secreted protein hMGRAP has been screened and cloned, and its biological function may specifically relate to its repeated glutamic acid sequence.     

Amino acid sequence of rat epidermal thiol proteinase inhibitor

The complete amino acid sequence of rat epidermal thiol proteinase inhibitor was determined. The unique 103-residue sequence was derived by analysis of two peptides generated by limited proteolysis of the native inhibitor with Staphylococcus aureus V8 protease and of three cyanogen bromide fragments. The protein has a high degree of sequence homology to either rat liver or human leucocyte inhibitor but is not identical and may represent a new type of low molecular weight thiol proteinase inhibitors.