cexe -mcherry fusion Search Results


cexe -mcherry fusion  (GenScript corporation)
90
GenScript corporation cexe -mcherry fusion
Unique Aat amino acid sequences were detected using PSI-BLAST. These sequences were using to identify the strains that encoded them in the NCBI identical protein groups. As the aat genes were present on contigs of whole genome sequencing projects it was not possible to assess if a strain encoded a gene on the chromosome or plasmid. Instead contigs were used to identify complete the complete Aat system. This analysis does not include strains that might encode the aat genes or <t>aap/cexE</t> on a separate genomic element. However, a total of 827 complete Aat systems were identified in the same nucleotide accession. The positions of these genes were used to assess the organisation of the aat operon. From this assessment five different classes of aat operon organisation were defined.
Cexe Mcherry Fusion, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cexe -mcherry fusion - by Bioz Stars, 2026-05
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Unique Aat amino acid sequences were detected using PSI-BLAST. These sequences were using to identify the strains that encoded them in the NCBI identical protein groups. As the aat genes were present on contigs of whole genome sequencing projects it was not possible to assess if a strain encoded a gene on the chromosome or plasmid. Instead contigs were used to identify complete the complete Aat system. This analysis does not include strains that might encode the aat genes or aap/cexE on a separate genomic element. However, a total of 827 complete Aat systems were identified in the same nucleotide accession. The positions of these genes were used to assess the organisation of the aat operon. From this assessment five different classes of aat operon organisation were defined.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Unique Aat amino acid sequences were detected using PSI-BLAST. These sequences were using to identify the strains that encoded them in the NCBI identical protein groups. As the aat genes were present on contigs of whole genome sequencing projects it was not possible to assess if a strain encoded a gene on the chromosome or plasmid. Instead contigs were used to identify complete the complete Aat system. This analysis does not include strains that might encode the aat genes or aap/cexE on a separate genomic element. However, a total of 827 complete Aat systems were identified in the same nucleotide accession. The positions of these genes were used to assess the organisation of the aat operon. From this assessment five different classes of aat operon organisation were defined.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Sequencing, Plasmid Preparation

(A) Culture supernatant of ETEC H10407 and aat mutants harbouring pCfaD grown in LB supplemented with L-arabinose. Cells were removed from the culture supernatant and remaining protein precipitated with trichloroacetic acid. Protein samples were separated by tris-tricine SDS-PAGE and stained with Coomassie or transferred to nitrocellulose for western blotting with polyclonal antibodies against CexE. (B) Whole cell lysates of the aat mutants and parental strain separated by tris-tricine SDS-PAGE. (C) Whole cell lysates of ETEC H10407 grown in LB with or without azide. (D) Whole cell lysates of EAEC 042, aap, aatD and aatD complemented strains grown in DMEM high glucose. The positions of uCexE, mCexE, uAap, mAap, and preCexE are indicated as appropriate. CexE and Aap were detected by western blotting (α-CexE and α-Aap, respectively) and RNA polymerase (α-RNAP) was included as a loading control where appropriate.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) Culture supernatant of ETEC H10407 and aat mutants harbouring pCfaD grown in LB supplemented with L-arabinose. Cells were removed from the culture supernatant and remaining protein precipitated with trichloroacetic acid. Protein samples were separated by tris-tricine SDS-PAGE and stained with Coomassie or transferred to nitrocellulose for western blotting with polyclonal antibodies against CexE. (B) Whole cell lysates of the aat mutants and parental strain separated by tris-tricine SDS-PAGE. (C) Whole cell lysates of ETEC H10407 grown in LB with or without azide. (D) Whole cell lysates of EAEC 042, aap, aatD and aatD complemented strains grown in DMEM high glucose. The positions of uCexE, mCexE, uAap, mAap, and preCexE are indicated as appropriate. CexE and Aap were detected by western blotting (α-CexE and α-Aap, respectively) and RNA polymerase (α-RNAP) was included as a loading control where appropriate.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: SDS Page, Staining, Western Blot, Control

His tagged CexE was isolated from cexE or cexE aatD double mutants harbouring pACYC- cexE -6His grown in the presence or absence of 17-ODYA and separated by SDS-PAGE. An azide linked Alexa Fluor 488 was conjugated to the alkyne moiety present in 17-ODYA by CuAAC. The incorporation of 17-ODYA into the target protein was detected by fluorescence (green bands) and the image was overlaid on the image of the SDS-PAGE gel. A known lipoprotein YraP was used as a positive control.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: His tagged CexE was isolated from cexE or cexE aatD double mutants harbouring pACYC- cexE -6His grown in the presence or absence of 17-ODYA and separated by SDS-PAGE. An azide linked Alexa Fluor 488 was conjugated to the alkyne moiety present in 17-ODYA by CuAAC. The incorporation of 17-ODYA into the target protein was detected by fluorescence (green bands) and the image was overlaid on the image of the SDS-PAGE gel. A known lipoprotein YraP was used as a positive control.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Isolation, SDS Page, Fluorescence, Positive Control

The membrane fraction of ETEC H10407 and aat mutant harbouring pCfaD were separated by SDS-PAGE. Transferred to nitrocellulose and probed for the cytoplasmic protein RNAP, lipoprotein NlpE and CexE using protein specific antibodies.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: The membrane fraction of ETEC H10407 and aat mutant harbouring pCfaD were separated by SDS-PAGE. Transferred to nitrocellulose and probed for the cytoplasmic protein RNAP, lipoprotein NlpE and CexE using protein specific antibodies.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Membrane, Mutagenesis, SDS Page

E. coli BL21(DE3) was transformed with pET26b, or pET26b- cexE and pACYCDuet-1 or pACYC- aatD . Cultures were grown in LB and protein production was stimulated with IPTG. Whole cell lysate samples were taken and separated by tris-tricine SDS-PAGE. CexE was detected using a polyclonal antibody. RNAP was used as a loading control. The position of mCexE and uCexE are indicated.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: E. coli BL21(DE3) was transformed with pET26b, or pET26b- cexE and pACYCDuet-1 or pACYC- aatD . Cultures were grown in LB and protein production was stimulated with IPTG. Whole cell lysate samples were taken and separated by tris-tricine SDS-PAGE. CexE was detected using a polyclonal antibody. RNAP was used as a loading control. The position of mCexE and uCexE are indicated.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Transformation Assay, SDS Page, Control

(A) AatD from EAEC 042 and ETEC H10407 were aligned with Lnt from MG1655. AatD sequences identified by PSI-BLAST were aligned and used to create a Weblogo. The catalytic residues of the C-N hydrolase family are highlighted. (B) The structure of Lnt compared to the predicted structure of ETEC H10407 AatD. The catalytic residues of Lnt and AatD are highlighted in orange. (C) Magnified view of the catalytic site of Lnt with the predicted structure of AatD superimposed. (D) Residues are numbered as they appear in Lnt. E. coli BL21(DE3) was producing CexE in the presence of each of the four single site substitution mutants of AatD. CexE was detected using a polyclonal antibody.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) AatD from EAEC 042 and ETEC H10407 were aligned with Lnt from MG1655. AatD sequences identified by PSI-BLAST were aligned and used to create a Weblogo. The catalytic residues of the C-N hydrolase family are highlighted. (B) The structure of Lnt compared to the predicted structure of ETEC H10407 AatD. The catalytic residues of Lnt and AatD are highlighted in orange. (C) Magnified view of the catalytic site of Lnt with the predicted structure of AatD superimposed. (D) Residues are numbered as they appear in Lnt. E. coli BL21(DE3) was producing CexE in the presence of each of the four single site substitution mutants of AatD. CexE was detected using a polyclonal antibody.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques:

(A) SignalP result of CexE sequence from ETEC H10407 (E3PPH2_ECOH1) (B) Weblogo of the 5 N-terminal residues of Aap and CexE sequences post Sec signal sequence cleavage. (C) ETEC H10407 cexE mutants transformed with pACYC184 (Δ cexE ) or pACYC- cexE -6His with either the wild-type sequence (WT cexE ) or one of the first five amino acids mutated to glutamic acid. CexE was detected by polyclonal antibodies and RNAP was used as a loading control. The percentage of mCexE was determined from 3 biological replicates.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) SignalP result of CexE sequence from ETEC H10407 (E3PPH2_ECOH1) (B) Weblogo of the 5 N-terminal residues of Aap and CexE sequences post Sec signal sequence cleavage. (C) ETEC H10407 cexE mutants transformed with pACYC184 (Δ cexE ) or pACYC- cexE -6His with either the wild-type sequence (WT cexE ) or one of the first five amino acids mutated to glutamic acid. CexE was detected by polyclonal antibodies and RNAP was used as a loading control. The percentage of mCexE was determined from 3 biological replicates.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Sequencing, Transformation Assay, Control

Aap and CexE sequences were aligned using ClustalO. The alignment was then placed into WebLogo.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Aap and CexE sequences were aligned using ClustalO. The alignment was then placed into WebLogo.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques:

(A) CexE-6His was isolated from a cexE mutant and a cexE aatD double mutant and subjected to LC-MS/MS. CexE and pro-CexE were trypsinated and separated by HPLC. (B) The indicated peak was subjected to MS/MS to identify the amino acid sequence.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: (A) CexE-6His was isolated from a cexE mutant and a cexE aatD double mutant and subjected to LC-MS/MS. CexE and pro-CexE were trypsinated and separated by HPLC. (B) The indicated peak was subjected to MS/MS to identify the amino acid sequence.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Isolation, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing

Heterologous acylation by AatD plasmid encoding mCherry with the CexE signal sequence followed by none, one (G), two (GG) or three (GGG) glycine residues post signal sequence was produced in an ETEC H10407 cexE mutant grown in the presence of 17-ODYA. The mCherry proteins were isolated using a C-terminal 8 His tag and azide linked Cy5 was incorporated into mCherry proteins using CuAAC. The acylation of mCherry was detected using fluorescence.

Journal: bioRxiv

Article Title: Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

doi: 10.1101/2020.09.23.310854

Figure Lengend Snippet: Heterologous acylation by AatD plasmid encoding mCherry with the CexE signal sequence followed by none, one (G), two (GG) or three (GGG) glycine residues post signal sequence was produced in an ETEC H10407 cexE mutant grown in the presence of 17-ODYA. The mCherry proteins were isolated using a C-terminal 8 His tag and azide linked Cy5 was incorporated into mCherry proteins using CuAAC. The acylation of mCherry was detected using fluorescence.

Article Snippet: The cexE -mCherry fusion and the aatD gene from ETEC H10407 were synthesised by GenScript.

Techniques: Plasmid Preparation, Sequencing, Produced, Mutagenesis, Isolation, Fluorescence