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 Collagen survival and its use for species identification in Holocene-lower Pleistocene bone fragments from British archaeological and paleontological sites

Antiqua ◽  
2011 ◽  
Vol 1 (1) ◽  
pp. 1 ◽  
Author(s):  
Mike Buckley ◽  
Matthew James Collins
Keyword(s):  
Species Identification ◽  
Collagen Type I ◽  
Systematic Investigation ◽  
Type I ◽  
Structural Protein ◽  
Collagen Peptides ◽  
Collagen Peptide ◽  
Peptide Mass ◽  
Lower Pleistocene ◽  
Bone Fragments

Proteins have long been known to persist in Quaternary bone fossils and are often targeted as a source of carbon used in radiocarbon dating and stable isotope analyses for determining provenance and obtaining dietary information. We have previously reported a technique using the dominant structural protein collagen (type I) as a source of genetic information for species identification in modern and relatively young (Holocene) archaeological samples. We report a systematic investigation of amino acid composition and collagen peptide mass fingerprints (PMF), for a range of samples dating back approximately 1.5 million years. Extrapolation from high temperature experimental decomposition rates predict that at a constant 10°C (the approximate mean annual air temperature in Britain today) it will take between 0.2 and 0.7 Ma for levels of collagen to fall to 1% of their original concentration in an optimal burial environment. Even when the glacial intervals of the British Quaternary are factored into the temperature calculations, the more conservative of these two estimates extends the range for collagen sequencing to the Lower Pleistocene as confirmed by the presence of collagen peptides in bones from the Weybourne Crag (~1.5 Ma). Collagen fingerprinting can extend the range of identifiable taxa present at sites with large assemblages of fragmentary bone material such as that encountered at the ~900 Ka site at Happisburgh (Norfolk, UK) recently identified as showing signs of the earliest humans in Britain.

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.

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 AP Collagen Peptides Prevent Cortisol-Induced Decrease of Collagen Type I in Human Dermal Fibroblasts

2021 ◽  
Vol 22 (9) ◽  
pp. 4788
Author(s):  
Minjung Chae ◽  
Il-Hong Bae ◽  
Sung Hwan Lim ◽  
Kyoungmi Jung ◽  
Jonghwa Roh ◽  
...  
Keyword(s):  
Stress Responses ◽  
Collagen Type ◽  
Transforming Growth Factor ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Human Dermal Fibroblasts ◽  
Collagen Peptides ◽  
Wide Range ◽  
Dependent Inhibition

Cortisol is an endogenous glucocorticoid (GC) and primary stress hormone that regulates a wide range of stress responses in humans. The adverse effects of cortisol on the skin have been extensively documented but the underlying mechanism of cortisol-induced signaling is still unclear. In the present study, we investigate the effect of cortisol on collagen type I expression and the effect of AP collagen peptides, collagen tripeptide-rich hydrolysates containing 3% glycine-proline- hydroxyproline (Gly-Pro-Hyp, GPH) from the fish skin, on the cortisol-mediated inhibition of collagen type I and the cortisol-induced signaling that regulates collagen type I production in human dermal fibroblasts (HDFs). We determine that cortisol downregulates the expression of collagen type I. AP collagen peptides or GC receptor (GR) inhibitors recover the cortisol-mediated inhibition of collagen type I and GR activation. AP collagen peptides or GR inhibitors also prevent the cortisol-dependent inhibition of transforming growth factor (TGF)-β signaling. AP collagen peptides or GR inhibitors are effective in the prevention of collagen type I inhibition mediated by cortisol in senescent HDFs and reconstituted human skin models. Taken together, GR signaling might be responsible for the cortisol-mediated inhibition of TGF-β. AP collagen peptides act as GR-mediated signaling blockers, preventing the cortisol-dependent inhibition of collagen type I. Therefore, AP collagen peptides have the potential to improve skin health.

scholarly journals Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea

1997 ◽  
Vol 323 (2) ◽  
pp. 557-565 ◽  
Author(s):  
Hao-Chi HSU ◽  
Chyong-Ing HSU ◽  
Rong-Hwa LIN ◽  
Chian-Liang KAO ◽  
Jung-Yaw LIN
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Protein Sequencing ◽  
Blue Staining ◽  
Volvariella Volvacea ◽  
Blocking Group ◽  
Calculated Molecular Mass ◽  
Fungal Immunomodulatory Protein ◽  
Immunomodulatory Protein ◽  
Single Polypeptide

A new fungal immunomodulatory protein (Fip) has been purified from the edible mushroom, Volvariella volvacea, and designated Fip-vvo. Analysis of the purified protein by SDS/PAGE followed by Coomassie Blue staining demonstrated that Fip-vvo is a single polypeptide with an apparent molecular mass of 15 kDa. Periodic acid/Schiff staining showed that this single polypeptide lacks carbohydrates. Using an in vitro bioassay measuring blast-formation stimulatory activity, Fip-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphocytes at a concentration of 5 μg/ml. Fip-vvo was capable of agglutinating rat red blood cells. Neither haemagglutination nor mitogenic activities were inhibited by mono- or dimeric sugars. In vivo, repeat administration of Fip-vvo greatly reduced the production of BSA-induced Arthus reaction in mice, whereas little effect was observed on the prevention of systemic anaphylaxis reactions. The selectively enhanced transcriptional expression of interleukin (IL)-2, IL-4, interferon-γ, tumour necrosis factor-α, lymphotoxin and IL-2 receptor by Fip-vvo was also demonstrated by reverse transcriptase-PCR. This finding suggests that Fip-vvo exerts its immunomodulatory effects via cytokine regulation. In addition, the complete amino acid sequence of Fip-vvo was obtained by direct protein sequencing. This protein consists of 112 amino acid residues with a blocked N-terminal end and has a calculated molecular mass of 12667 Da not including the N-terminal blocking group. By gel filtration analysis, Fip-vvo exhibited a molecular mass of 26 kDa for the native molecules in PBS. This result indicates that native Fip-vvo is most likely a non-covalently associated homodimeric molecule.

scholarly journals Purification and characterization of a connective-tissue-degrading metalloproteinase from the cytosol of metastatic melanoma cells

1987 ◽  
Vol 245 (2) ◽  
pp. 429-437 ◽  
Author(s):  
S Zucker ◽  
T Turpeenniemi-Hujanen ◽  
N Ramamurthy ◽  
J Wieman ◽  
R Lysik ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Type I Collagen ◽  
Collagen Type I ◽  
Enzymic Activity ◽  
Affinity Column ◽  
Type I ◽  
Type Iv ◽  
Affinity Column Chromatography ◽  
Molecular Masses

A metalloproteinase with activity against type IV collagen, type I collagen and gelatin has been purified from the cytosol of a highly metastatic mouse melanoma by anion-exchange, zinc-chelated and lectin-affinity column chromatography. The purified enzyme has a molecular mass of approx. 59 kDa and on isoelectric focusing in two-dimensional gels produced three spots with apparent isoelectric points (pI) between 5.7 and 6.1. Enzymic activity with collagen, but not gelatin, substrates was latent, requiring activation by trypsin or organomercurials. Trypsin activation of this metalloproteinase was accompanied by a change in molecular mass, whereas autoactivation after 1 month's storage, was not. The degradation of types I and IV collagen by the melanoma enzyme yielded products of lower molecular masses than those yielded by mammalian collagenases, this characteristic thus differentiating this metalloproteinase from classical collagenases.

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