Isolation and characterization of a gene encoding a chymotrypsin-like serine protease fromStreptomyces lividans66

1996 ◽  
Vol 42 (3) ◽  
pp. 284-288 ◽  
Author(s):  
Craig Binnie ◽  
Linda Liao ◽  
Eva Walczyk ◽  
Lawrence T. Malek
Keyword(s):  
Amino Acids ◽  
Serine Protease ◽  
Streptomyces Griseus ◽  
Nucleotide Sequence Analysis ◽  
Predicted Amino Acid Sequence ◽  
Gene Encoding ◽  
Hybridization Probe ◽  
Putative Gene ◽  
Isolation And Characterization ◽  
High Degree

A gene encoding a Streptomyces lividans homologue of the chymotrypsin-like serine protease (SAM-P20) of Streptomyces albogriseolus was isolated using the Streptomyces griseus prtB gene as a hybridization probe. Nucleotide sequence analysis of a representative clone uncovered the possible presence of a sequence of 900 nucleotides encoding 300 amino acids, including a putative "prepro" region of 115 amino acids. Alignment of the predicted amino acid sequence of this putative gene with other members of the family of Streptomyces extracellular chymotrypsin-like proteases indicated a high degree of homology in all cases, especially with the SAM-P20 protease. This gene product has been identified as the second member of a potentially larger family of SAL (SAM-P20-like) proteases in S. lividans 66. Keywords: Streptomyces, protease, chymotrypsin.

Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein

1992 ◽  
Vol 38 (3) ◽  
pp. 193-202 ◽  
Author(s):  
Angus Gilchrist ◽  
James A. Fisher ◽  
John Smit
Keyword(s):  
Amino Acids ◽  
Surface Layer ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Caulobacter Crescentus ◽  
Amino Acid Profile ◽  
Nucleotide Sequence Analysis ◽  
Predicted Amino Acid Sequence ◽  
Gene Encoding ◽  
Methionine Residue

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98 132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest. Key words: Caulobacter crescentus, surface layer, nucleotide sequence, rsaA, calcium.

Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene

1983 ◽  
Vol 3 (2) ◽  
pp. 203-213
Author(s):  
J M Pipas ◽  
K W Peden ◽  
D Nathans
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Carboxyl Terminus ◽  
Nucleotide Sequence Analysis ◽  
Morphological Transformation ◽  
Isolation And Characterization

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.

Characterization of the gene encoding the glutamic-acid-specific protease of Streptomyces griseus

1993 ◽  
Vol 71 (9-10) ◽  
pp. 454-461 ◽  
Author(s):  
Sachdev S. Sidhu ◽  
Gabriel B. Kalmar ◽  
Thor J. Borgford
Keyword(s):  
Glutamic Acid ◽  
Serine Protease ◽  
Streptomyces Griseus ◽  
Gene Encoding ◽  
Lysobacter Enzymogenes ◽  
Glutamic Acid Residue ◽  
Specific Protease ◽  
Mature Enzyme ◽  
Additional Segment ◽  
Pro Region

The complete gene sequence (sprE) for the glutamic-acid-specific serine protease (SGPE) of the gram-positive bacterium Streptomyces griseus is reported. The sprE gene encodes a 355 amino acid pre–pro–mature enzyme. The presence of a glutamic acid residue at the junction of the pro and mature segments of the protein suggests that the enzyme is self-processing. SGPE was found to have extensive homology with the S. griseus proteases A and B. However, there is an additional segment to the pro region of SGPE, lacking in proteases A and B, which has significant homology to the pro region of the α-lytic protease of the gram-negative bacterium Lysobacter enzymogenes. Expression of recombinant SGPE in Bacillus subtilis is also reported, and the enzyme is shown to be self-processing.Key words: serine protease, maturation, expression, propeptide.

scholarly journals Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T-antigen gene.

1983 ◽  
Vol 3 (2) ◽  
pp. 203-213 ◽  
Author(s):  
J M Pipas ◽  
K W Peden ◽  
D Nathans
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Carboxyl Terminus ◽  
Nucleotide Sequence Analysis ◽  
Morphological Transformation ◽  
Isolation And Characterization

A series of mutants of simian virus 40 has been constructed with deletions in the coding sequence for large T antigen. Nucleotide sequence analysis indicates that 4 mutants have in-phase and 11 have out-of-phase deletions. Mutant DNAs were assayed for the following activities: the ability to form plaques, the ability to produce T antigen as scored by indirect immunofluorescence, viral DNA replication, and morphological transformation of rat cells. Two viable mutants were found, and these had deletions confined to the carboxyl terminus of T antigen. Only those mutants coding for polypeptides greater than 40% of the length of wildtype T antigen produced detectable nuclear fluorescence. The two viable mutants with deletions in the carboxyl terminus of the protein retained the ability both to replicate their DNA, although at a reduced level, and to transform nonpermissive cells. Mutants with sequence changes that result in the loss of more than 117 amino acids from the carboxyl terminus were not viable and were also defective in the DNA replication and transformation functions of T antigen, although several produced detectable nuclear fluorescence. These functions were also sensitive to the removal of amino acids near the amino terminus and in the middle of the protein.

Isolation and Characterization of the Cryptococcus neoformans MATa Pheromone Gene

Genetics ◽  
2002 ◽  
Vol 160 (3) ◽  
pp. 935-947
Author(s):  
Carol M McClelland ◽  
Jianmin Fu ◽  
Gay L Woodlee ◽  
Tara S Seymour ◽  
Brian L Wickes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Cryptococcus Neoformans ◽  
Mating Types ◽  
Filament Formation ◽  
Gene Encoding ◽  
Conserved Sequence ◽  
Isolation And Characterization ◽  
Conserved Genes

Abstract Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATα. The mating pathway of this fungus has a number of conserved genes, including a MATα-specific pheromone (MFα1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATα cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfα1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFα1 gene, which is found in MATα strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.

scholarly journals Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin

2011 ◽  
Vol 79 (6) ◽  
pp. 2440-2450 ◽  
Author(s):  
Shaper Mirza ◽  
Landon Wilson ◽  
William H. Benjamin ◽  
Jan Novak ◽  
Stephen Barnes ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptococcus Pneumoniae ◽  
Serine Protease ◽  
Human Lactoferrin ◽  
Free Form ◽  
Capsular Type ◽  
Gene Encoding ◽  
Content Type ◽  
Killing Activity

ABSTRACTIt is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, includingStreptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease,prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P< 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin killsStreptococcus pneumoniaerequires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

Sign in / Sign up

Export Citation Format

Share Document