scholarly journals Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase

1994 ◽  
Vol 300 (1) ◽  
pp. 85-90 ◽  
Author(s):  
B Verjans ◽  
F De Smedt ◽  
R Lecocq ◽  
V Vanweyenberg ◽  
C Moreau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Bovine Brain ◽  
Cloning And Expression ◽  
Major Protein ◽  
Type I ◽  
Degenerate Primers ◽  
Calculated Molecular Mass ◽  
Major Protein Band ◽  
Inositol 1,4,5 Trisphosphate

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

scholarly journals Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

2003 ◽  
Vol 69 (1) ◽  
pp. 162-169 ◽  
Author(s):  
Naoki Tsuruoka ◽  
Toru Nakayama ◽  
Masako Ashida ◽  
Hisashi Hemmi ◽  
Masahiro Nakao ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Molecular Mass ◽  
Gene Cloning ◽  
Collagen Type I ◽  
Protein Sequencing ◽  
Collagenolytic Activity ◽  
Efficient Production ◽  
Type I ◽  
Calculated Molecular Mass ◽  
Collagen Peptides

ABSTRACT Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k cat, 5.41 s−1; Km , 32 μM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k cat, 351 s−1; Km , 214 μM), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.

scholarly journals Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase

1990 ◽  
Vol 272 (1) ◽  
pp. 107-112 ◽  
Author(s):  
K Takazawa ◽  
J Vandekerckhove ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Rat Brain ◽  
Kinase Activity ◽  
Limited Proteolysis ◽  
Cloning And Expression ◽  
Terminal Part ◽  
Dependent Protein Kinase ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Protein

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx. 50,000 on SDS/polyacrylamide gels. Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000. Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive. A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library. The 5′ end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase. The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II. When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity. Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.

scholarly journals A statherin and calcium enriched layer at the air interface of human parotid saliva

2005 ◽  
Vol 389 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Gordon B. PROCTOR ◽  
Sawsan HAMDAN ◽  
Guy H. CARPENTER ◽  
Peter WILDE
Keyword(s):  
Surface Layer ◽  
Molecular Mass ◽  
Calcium Binding ◽  
Solid Phase ◽  
Salivary Protein ◽  
Major Protein ◽  
Parotid Saliva ◽  
Surface Rheology ◽  
Major Protein Band ◽  
The Mean

Parotid saliva placed in 35-mm-diameter tissue culture dishes developed increasing surface viscoelasticity at the interface with air. A surface layer became visible with time, and was collected and analysed by protein electrophoresis which indicated that a single protein (pI 4.2; molecular mass approx. 6 kDa) predominated. Western blot analysis demonstrated that the major protein band reacted with an antiserum directed against the C-terminal of the calcium-binding salivary protein statherin. Matrix-assisted laser-desorption ionization–time-of-flight MS analysis gave a molecular mass of 5380 Da for the protein, corresponding to the molecular mass of statherin. Staining of film protein in electrophoresis gels was compared with statherin synthesized on a solid phase, and the mean statherin content of film formed from 1 ml of parotid saliva was measured as 7 nmol. The mean calcium content of the surface layer was 250 nmol. Surface rheology was greatly decreased in the presence of EDTA, whereas surface tension of saliva was unaffected by calcium chelation, suggesting that protein accumulated at the surface was unaffected. The results suggest that a layer rich in statherin forms at the interface of saliva and air, and that the surface rheology developed is dependent upon protein interactions mediated by calcium. The surface layer may enhance the function of saliva as a protective layer on the mucosal surfaces and teeth.

Molecular Cloning and Expression of β-1,4-endoxylanase Gene from Chaetomium cupreum in Yeast Saccharomyces cerevisiae

2013 ◽  
Vol 779-780 ◽  
pp. 191-194
Author(s):  
Hai Yan Zhang ◽  
Wen Rao Li ◽  
Min Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Heterologous Expression ◽  
Molecular Mass ◽  
Molecular Cloning ◽  
Cloning And Expression ◽  
Cdna Fragment ◽  
Gene Encoding ◽  
Calculated Molecular Mass

The gene encoding an endo-β-1,4-xylanase (XynCC) fromchaetomium cupreumwas amplified using PCR. The nucleotide sequence of a 690 bp cDNA fragment was determined. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 24.7 kDa. The XynCC gene was inserted into the pYES2 vector and transferred into the cells ofS. cerevisiaeH158 for heterologous expression.

197 MOLECULAR CLONING AND EXPRESSION OF THE CASHMERE GOAT IZUMO GENE

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
R. H. Na ◽  
L. Liang ◽  
L. Fu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Protein ◽  
Northern Blotting ◽  
Cloning And Expression ◽  
Immunoglobulin Superfamily ◽  
Predicted Amino Acid Sequence ◽  
Type I ◽  
Cashmere Goat ◽  
Calculated Molecular Mass

During the fertilization process, complex events are involved in the fusion between the reacted spermatozoa and the mature oocyte. Fusion implies that many proteins are present on the cell membrane of the gametes. Recently, a new protein, Izumo, has been shown to play a role in the sperm–egg fusion. Izumo was identified through the generation of a monoclonal antibody that inhibits this fusion. This protein belongs to the immunoglobulin superfamily type I transmembrane protein. Izumo can be detected only after acrosome reaction at the spermatozoal surface. The cashmere goat Izumo gene was identified and cloned by 3′ and 5′ rapid amplification of cDNA ends-PCR. The expression of cashmere goat Izumo was examined by RT-PCR and Northern blotting. The full-length cDNA of cashmere goat Izumo contains 1536 bp and an open reading frame of 1035 bp, encoding a polypeptide of 344 amino acids with a calculated molecular mass of 38.76 kDa and a theoretical isoelectric point of 8.18. This predicted amino acid sequence showed 89.82% amino acid identity with the bovine Izumo. The deduced amino acid sequence contained 3 conserved domains of the signal peptide, immunoglobulin-like domain, and transmembrane region. Reverse transcription-PCR and Northern blotting analysis showed that cashmere goat Izumo transcripts were highly expressed in the testis, caput, corpus, cauda epididymis. Cashmere goat Izumo may play a role in the biological process of fertilization. This work was supported by the National Natural Science Foundation (No. 30560103 and No.30740043), China, and the China Postdoctoral Science Foundation.

scholarly journals The endolysins of bacteriophages CMP1 and CN77 are specific for the lysis of Clavibacter michiganensis strains

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2366-2373 ◽  
Author(s):  
Johannes Wittmann ◽  
Rudolf Eichenlaub ◽  
Brigitte Dreiseikelmann
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Molecular Mass ◽  
Host Range ◽  
High Specificity ◽  
Clavibacter Michiganensis ◽  
Calculated Molecular Mass ◽  
Bacteriolytic Activity

Putative endolysin genes of bacteriophages CMP1 and CN77, which infect Clavibacter michiganensis subsp. michiganensis and C. michiganensis subsp. nebraskensis, respectively, were cloned and expressed in Escherichia coli. The His-tagged endolysin of CMP1 consists of 306 amino acids and has a calculated molecular mass of 34.8 kDa, while the His-tagged endolysin of CN77 has 290 amino acids with a molecular mass of 31.9 kDa. The proteins were purified and their bacteriolytic activity was demonstrated. The bacteriolytic activity of both enzymes showed a host range which was limited to the respective C. michiganensis subspecies and did not affect other bacteria, even those closely related to Clavibacter. Due to the high specificity of the CMP1 and CN77 endolysins they may be useful tools for biocontrol of plant-pathogenic C. michiganensis without affecting other bacteria in the soil.

scholarly journals Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung

1998 ◽  
Vol 330 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Charles Mark ENSOR ◽  
Hongxing ZHANG ◽  
Hsin-Hsiung TAI
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Leukotriene B4 ◽  
Cloning And Expression ◽  
Library Screening ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Apparent Homogeneity ◽  
Pcr Product ◽  
Cloned Cdna

15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent homogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating that the enzyme is a monomer. 15-Oxo-PGE1, 15-oxo-PGE2 and 15-oxo-PGF2α were found to be substrates for the enzyme, whereas the corresponding 15-hydroxyprostaglandins were not. The reverse reaction, the oxidation of 13,14-dihydro-15-oxo-PGE1 to 15-oxo-PGE1, was not observed. Either NADH or NADPH could serve as a coenzyme. However, the Vmax with NADH was approx. 3-fold that with NADPH, while the Km for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was achieved by PCR and library screening. A 600 bp PCR product containing the sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cDNA clone that contained the entire PGR coding sequence of 987 bp was obtained. The sequence codes for a protein of 329 amino acid residues with a calculated molecular mass of 35791 Da. Homology analysis indicated that the sequence is virtually identical with that of leukotriene B4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izumi and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of this cDNA in Escherichia coli resulted in a protein exhibiting both PGR and LTB4 12-hydroxydehydrogenase activities. However, the specific activity of PGR with 15-oxo-PGE1 as a substrate was approx. 300-fold that of LTB4 12-hydroxydehydrogenase. These results indicate that the cloned cDNA codes for a protein with two different enzyme activities, with 15-oxoprostaglandins as the preferred substrates.

scholarly journals Purification and characterization of rat epididymal-fluid α-d-mannosidase: similarities to sperm plasma-membrane α-d-mannosidase

1993 ◽  
Vol 290 (2) ◽  
pp. 427-436 ◽  
Author(s):  
D R P Tulsiani ◽  
M D Skudlarek ◽  
S K Nagdas ◽  
M C Orgebin-Crist
Keyword(s):  
Molecular Mass ◽  
Plasma Membranes ◽  
Antigenic Site ◽  
Protein Band ◽  
Soluble Form ◽  
Major Protein ◽  
Major Protein Band ◽  
Membrane Bound ◽  
Epididymal Fluid

We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.

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