scholarly journals Antigenic and Molecular Conservation of the Gonococcal NspA Protein

1999 ◽  
Vol 67 (6) ◽  
pp. 2855-2861 ◽  
Author(s):  
Martin Plante ◽  
Nathalie Cadieux ◽  
Clément R. Rioux ◽  
Josée Hamel ◽  
Bernard R. Brodeur ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Protein A ◽  
Surface Protein ◽  
Meningococcal Infection ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Intact Cells ◽  
Reading Frame ◽  
Calculated Molecular Weight

ABSTRACT A low-molecular-weight protein named NspA (neisserial surface protein A) was recently identified in the outer membrane of allNeisseria meningitidis strains tested. Antibodies directed against this protein were shown to protect mice against an experimental meningococcal infection. Hybridization experiments clearly demonstrated that the nspA gene was also present in the genomes of the 15 Neisseria gonorrhoeae strains tested. Cloning and sequencing of the nspA gene of N. gonorrhoeaeB2 revealed an open reading frame of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a calculated molecular weight of 18,316 and a pI of 10.21. Comparison of the predicted amino acid sequence of the NspA polypeptides from the gonococcal strains B2 and FA1090, together with that of the meningococcal strain 608B, revealed an identity of 93%, suggesting that the NspA protein is highly conserved among pathogenic Neisseria strains. The level of identity rose to 98% when only the two gonococcal predicted NspA polypeptides were compared. To evaluate the level of antigenic conservation of the gonococcal NspA protein, monoclonal antibodies (MAbs) were generated. Four of the seven NspA-specific MAbs described in this report recognized their corresponding epitope in 100% of the 51 N. gonorrhoeae strains tested. Radioimmunobinding assays clearly indicated that the gonococcal NspA protein is exposed at the surface of intact cells.

scholarly journals Characterization of cry1Cb3 and cry1Fb7 from Bacillus thuringiensis subsp. galleriae

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Tianpei Huang ◽  
Ying Xiao ◽  
Jieru Pan ◽  
Lingling Zhang ◽  
Ivan Gelbič ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Bacillus Thuringiensis Subsp ◽  
Calculated Molecular Weight ◽  
Pcr Rflp ◽  
Genes Encoding

AbstractTwo cry1-type genes encoding insecticidal crystal proteins (ICPs) were detected by PCR-RFLP and cloned from Bacillus thuringiensis subsp. galleriae 87. The nucleotide sequences were deposited in GenBank with accession numbers EU679501 and EU679502, and designated as cry1Fb7 and cry1Cb3 respectively by B. thuringiensis Delta- Endotoxin Nomenclature Committee. cry1Cb3 shared 99% homology with other cry1Cb genes. The existence of two additional stop codons indicated cry1Cb3 was a silent gene. The cry1Cb3 was 3531 bp with 38.98% G+C content and its first open reading frame (ORF) encoded a protein of 213 amino acid residues with a calculated molecular weight of 23.8 kDa and a predicted pI value of 4.63. Five amino acid sequence blocks (block 1, block 2, block 3, block 4 and block 5) were found in Cry1Cb3. Translation of cry1Fb7 revealed an ORF of 3525 bp with 39.12% G+C content and a protein with a calculated molecular weight of 133.2 kDa and a predicted pI value of 5.18. Cry1Fb7 had five amino acid sequence blocks (blocks 1, 2, 3, 4 and 5) and three domains (I, II and III), which consisted of 218 residues (Leu

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

Molecular cloning and hormonal control in the ovary of connexin 31.5 mRNA and correlation with the appearance of oocyte maturational competence in red seabream

2000 ◽  
Vol 203 (21) ◽  
pp. 3299-3306
Author(s):  
C.Y. Choi ◽  
F. Takashima
Keyword(s):  
Amino Acid ◽  
Hormonal Control ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Red Seabream ◽  
Consensus Sequences ◽  
Cdna Sequences ◽  
Connexin Gene ◽  
Connexin Genes

Gap junctions are aggregates of intercellular channels, composed of the protein connexin (Cx), between adjacent cells. This study examined whether, in the ovary of the red seabream Pagrus major, the connexin gene essential for the production of RNA and protein during the acquisition of oocyte maturational competence is active. Mixed primers for this reaction were designed on the basis of the high sequence homology of selected regions of known connexin genes. Polymerase-chain-reaction-amplified cDNA fragments generated by 3′ and 5′ rapid amplication of cDNA ends were combined to generate full-length cDNA sequences. The resulting 2400 base pair cDNA had an open reading frame encoding a polypeptide containing 275 amino acid residues (31493 Da; Cx31.5). Hydropathicity analysis of the predicted amino acid sequence indicated that red seabream Cx31.5 has four major hydrophobic regions and four major hydrophilic regions indicative of a topology similar to that of known connexins. Typical connexin consensus sequences were also observed in the first and second extracellular loops. During the acquisition of oocyte maturational competence, red seabream Cx31.5 mRNA transcription levels increased after treatment with gonadotropin-II. It is therefore proposed that expression of Cx31.5 contributes to the acquisition of oocyte maturational competence in this species.

scholarly journals Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

1987 ◽  
Vol 7 (10) ◽  
pp. 3409-3417 ◽  
Author(s):  
A Saxena ◽  
R Padmanabha ◽  
C V Glover
Keyword(s):  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Protein Kinase ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Open Reading Frame ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
Reading Frame

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.

scholarly journals Cloning and Characterization of a Cryptic Haloacid Dehalogenase from Burkholderia cepacia MBA4

1999 ◽  
Vol 181 (19) ◽  
pp. 6003-6009 ◽  
Author(s):  
Jimmy S. H. Tsang ◽  
Laiju Sam
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Burkholderia Cepacia ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Haloacid Dehalogenase ◽  
Reading Frame ◽  
Relative Molecular Weight

ABSTRACT Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards thel-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.

Determination of Low Molecular weight Immunogenic Omp28 Protein and Gene of Salmonella typhimurium

2019 ◽  
Vol 8 (5) ◽  
pp. 172-177
Author(s):  
Rajeev Kumar ◽  
S. P. Singh ◽  
Mahesh Kumar ◽  
Anil Kumar
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Outer Membrane ◽  
Close Agreement ◽  
Outer Membrane Proteins ◽  
Low Molecular Weight ◽  
Predicted Amino Acid Sequence ◽  
Calculated Molecular Weight ◽  
Amplicon Size

Outer membrane of Gram-negative bacteria has complex profile of proteins. The outer membrane proteins (OMPs) isolated from S. typhimurium by urea-EDTA extraction method and analysed through SDS-PAGE showed a complex electrophoretic profile having more than 15 low molecular weight proteins with molecular masses ranging between 3.5 and 43 kDa. The most important outer membrane protein (Omp28) of S. typhimurium with submolecular masses of 12.32kDa of main protein was recovered. The gene responsible for expression of this protein was also amplified through PCR and sequenced, showed 341bp amplicon size and predicted amino acid sequence of this pro-tein was determined. The Antigenic index was calculated from amino acid sequence of same gene and found 2.2 (0.1-2.2) suggesting highly antigenic in nature. The experimentally determined values are close agreement with the theoretically calculated molecular weight 12.32 kDa and pI: 9.61 from the gene sequence of this protein. The antigenic natures of predicted protein values are close agreement with experimental determent of Omp28 of S. typhimurium a possible formula for vaccine developmental of genus Salmonella.

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II

1987 ◽  
Vol 7 (10) ◽  
pp. 3409-3417
Author(s):  
A Saxena ◽  
R Padmanabha ◽  
C V Glover
Keyword(s):  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Protein Kinase ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Open Reading Frame ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
Reading Frame

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation

1985 ◽  
Vol 5 (10) ◽  
pp. 2684-2696
Author(s):  
D H Smith ◽  
D M Kegler ◽  
E B Ziff
Keyword(s):  
Amino Acid ◽  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Acid Protein ◽  
Dna Replication Origin ◽  
E1a Protein

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

The C-terminal sequence of amino acid residues of κ-casein isolated from buffalo's milk

1967 ◽  
Vol 34 (1) ◽  
pp. 85-88 ◽  
Author(s):  
M. H. Abd El-Salam ◽  
W. Manson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Chain ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Carboxypeptidase A ◽  
Phosphorous Content ◽  
Single Peptide

SummaryWhen κ-casein from buffalo's milk was treated with carboxypeptidase A (EC 3. 4. 2. 1),4 amino acids, valine, threonine, serine and alanine were released from the protein in a manner consistent with the view that they originate in the C-terminal sequence of a single peptide chain. The amounts produced suggest a minimum molecular weight for buffalo κ-casein of approximately 17000, in agreement with the value calculated from the phosphorous content on the basis of the presence of 2 phosphorus atoms/molecule. A comparison is made with the C-terminal sequence reported for bovine κ-casein.

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