Screening for novel immungenic proteins of Klebsiella pneumoniae MGH 78578
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ABSTRACT:
ORGANISM(S): Klebsiella pneumoniae
PROVIDER: GSE52536 | GEO | 2014/02/02
SECONDARY ACCESSION(S): PRJNA229171
REPOSITORIES: GEO
Similar Datasets
Project description:The screening of a cDNA derived expression library of Klebsiella pneumoniae MGH 78578 expressed in E.coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (ompA, mdh) and negative (pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from K. pneumoniae by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 14 novel immunogenic proteins could be identified. In total 1536 (4 x 384) different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: ompA1, ompA2 and mdh (3 x 40 replicates) as positive reference proteins, as they have been described as immunogenic before. gapA and pyrC (2 x 40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 376 different samples and 8 controls. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates). For identification rabbit polyclonal antibody to K. pneumoniae (Acris AP00792PU-N) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.
2014-02-02 | E-GEOD-52536 | biostudies-arrayexpress
Project description:The screening of a cDNA derived expression library of Salmonella Enteritidis 125109 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (fimA) and negative (argC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from S. Enteritidis by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 9 novel immunogenic proteins could be identified. In total 1536 different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: fimA from 3 different samples (40 replicates) as positive reference proteins, as it has been described as immunogenic before. GapA from Klebsiella pneumoniae and Campylobacter jejuni (40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 384 different samples. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates each). For identification rabbit polyclonal antibody to S. enterica (Abcam ab35156) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.
2013-10-01 | E-GEOD-49605 | biostudies-arrayexpress
Project description:Screening of 14 novel proteins derived from Klebsiella pneumoniae MGH 78578 identified prior via screening of cDNA libraries. The full-length proteins were attached using a specific HaloTag to their corresponding ligand surface, HaloLink. Screening was performed using two different polyclonal antibodies to Klebsiella pneumoniae (Acris AP00792PU-N and Abcam ab20947) and detection achieved by Goat polyclonal to rabbit IgG conjugated with Chromeo-546 (Abcam ab60317). In order to assess their potential immungenic nature and rank the proteins investigated, comparative analysis using already described antigens from K. pneumoniae were used in the assay. Each microarray was seperated into different incubation chambers using the 16-well ProPlate (Grace Biolabs) multi-well gaskets. As positive references ompA and mdh were used. For negative control gapA was used and the crude lysates of the expression host (Acella E.coli) and buffer were spotted as well.Samples and controls were spotted with five replicates each. Incubation was performed using different antibodies reactive to K. pneumoniae.
2014-02-02 | E-GEOD-52538 | biostudies-arrayexpress
Project description:The screening of a cDNA derived expression library of Salmonella Enteritidis 125109 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (fimA) and negative (argC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from S. Enteritidis by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 9 novel immunogenic proteins could be identified.
2013-10-01 | GSE49605 | GEO
Project description:The screening of a cDNA derived expression library of Campylobacter jejuni NCTC 11168 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. 1536 different clones were screened including positive (hisJ, cjaA, peb1a) and negative (argC, pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from C. jejuni by selecting clones showing a high signal intensity in comparison to the known antigens used as positive markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein, and these proteins were then investigated further. Consequently, 22 novel immunogenic proteins could be identified.
2013-04-06 | GSE45268 | GEO
Project description:Sequences of 11 amino acids belonging to the KPN_00363 protein and KPN_00459 protein from Klebsiella pneumoniae MGH 78578 which was previously identified as potentially immunogenic was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence.
2014-08-22 | GSE60588 | GEO
Project description:The screening of a cDNA derived expression library of Campylobacter jejuni NCTC 11168 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. 1536 different clones were screened including positive (hisJ, cjaA, peb1a) and negative (argC, pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from C. jejuni by selecting clones showing a high signal intensity in comparison to the known antigens used as positive markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein, and these proteins were then investigated further. Consequently, 22 novel immunogenic proteins could be identified. In total, 1536 (4 x 384) different lysates were spotted on different microarray slides. Each slide contained 3600 distinct spots, separated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: hisJ, cjaA and peb1 (3 x 40 replicates) as positive reference proteins, as they have been described as immunogenic before; argC and pyrC (2 x 40 replicates) as negative reference proteins; two sets of E. coli cell lysates without fusion proteins expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates); and a buffer control (24 replicates). Therefore, each set of replicate slides contained 376 different samples and 8 controls. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates each). For identification, rabbit polyclonal antibody to C. jejuni (Acris AP24002PU-N) as primary and goat polyclonal to rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards, both compartments were incubated with secondary antibody.
2013-04-06 | E-GEOD-45268 | biostudies-arrayexpress
Project description:To investigate the whole-genome gene expression difference between the wild-type and capsule deletion mutant in Klebsiella pneumoniae MGH 78578. The mutants analyzed in this study are further described in Huang T.W., Stapleton J.C., Chang H.Y., Tsai S.F., Palsson B.O., Charusanti P. Capsule removal via lambda-Red knockout system perturbs biofilm formation and fimbriae extression in Klesiella pneumoniae MGH 78578 (manuscript submission)
2013-12-31 | GSE40011 | GEO
Project description:To investigate the whole-genome gene expression difference between the wild-type and capsule deletion mutant in Klebsiella pneumoniae MGH 78578. The mutants analyzed in this study are further described in Huang T.W., Stapleton J.C., Chang H.Y., Tsai S.F., Palsson B.O., Charusanti P. Capsule removal via lambda-Red knockout system perturbs biofilm formation and fimbriae extression in Klesiella pneumoniae MGH 78578 (manuscript submission) A six chip study using total RNA recovered from three separate wild-type cultures and three separate cultures of a capsule deltion mutant of Klebsiella pneumoniae MGH 78578. The capsule gene cluster (KPN_02493 to KPN_02515) was entirely removed in the capsule deletion mutant. Each chip measures the expression level of 5,305 genes from Klebsiella pneumoniae MGH 78578 and the associated five plasmids (pKPN3, pKPN4, pKPN5, pKPN6 and pKPN7) with 50-mer oligo tiling array with 30-mer spacer.
2013-12-31 | E-GEOD-40011 | biostudies-arrayexpress
Project description:Screening of 14 novel proteins derived from Klebsiella pneumoniae MGH 78578 identified prior via screening of cDNA libraries. The full-length proteins were attached using a specific HaloTag to their corresponding ligand surface, HaloLink. Screening was performed using two different polyclonal antibodies to Klebsiella pneumoniae (Acris AP00792PU-N and Abcam ab20947) and detection achieved by Goat polyclonal to rabbit IgG conjugated with Chromeo-546 (Abcam ab60317). In order to assess their potential immungenic nature and rank the proteins investigated, comparative analysis using already described antigens from K. pneumoniae were used in the assay.
2014-02-02 | GSE52538 | GEO