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.

Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp60 protein, BA60K

1992 ◽  
Vol 12 (1) ◽  
pp. 47-62 ◽  
Author(s):  
R.Martin Roop ◽  
Michelle L. Price ◽  
Bruce E. Dunn ◽  
Stephen M. Boyle ◽  
Nammalwar Sriranganathan ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Molecular Cloning ◽  
Brucella Abortus ◽  
Nucleotide Sequence Analysis ◽  
Gene Encoding ◽  
Hsp60 Protein

scholarly journals Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 739-752 ◽  
Author(s):  
C A Woolford ◽  
C K Dixon ◽  
M F Manolson ◽  
R Wright ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Open Reading Frame ◽  
Relative Molecular Mass ◽  
Cell Fractionation ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

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.

Molecular cloning and determination of the nucleotide sequence of a gene encoding salt-tolerant glutaminase from Micrococcus luteus K-3

1996 ◽  
Vol 82 (6) ◽  
pp. 592-597 ◽  
Author(s):  
Mamoru Wakayama ◽  
Yoshio Nagano ◽  
Nandakumar Renu ◽  
Tatsuro Kawamura ◽  
Kenji Sakai ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Molecular Cloning ◽  
Micrococcus Luteus ◽  
Salt Tolerant ◽  
Gene Encoding
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