sgkeap1  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: Establishing the covalently reactive peptide/protein pair SnoopTag and SnoopCatcher. (A) Spontaneous isopeptide bond formation between Lys and Asn, releasing ammonia. (B) Cartoon of splitting RrgA D4 domain [based on Protein Data Bank (PDB) ID code 2WW8] to make SnoopTag and SnoopCatcher. Reactive residues are in cyan. (C) SnoopTag-MBP reaction with SnoopCatcher, each at 10 µM, after 2 h at 25 °C analyzed by SDS/PAGE with Coomassie staining, alongside controls with Ala mutation of SnoopTag’s reactive Lys (KA) or SnoopCatcher’s reactive Asn (NA). (D) Isopeptide bond formation between SnoopTag peptide and SnoopCatcher shown by mass spectrometry. (E) Time course of SnoopTag reaction with a 1:1 or 2:1 ratio of SnoopCatcher to SnoopTag-MBP, tested as in C. (F) Time course of SnoopCatcher reaction with a 1:1, 2:1, or 4:1 ratio of SnoopTag-MBP to SnoopCatcher, tested as in C. Error bars are mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Mutagenesis, Mass Spectrometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: SnoopTag and SnoopCatcher reaction conditions. (A) Point mutations to make SnoopCatcher. From the C-terminal domain of RrgA in cartoon format (based on PDB ID code 2WW8), residues mutated are shown in space-fill with carbons in cyan (G842 was changed to T and D848 to G). The Lys, Asn, and Glu involved in isopeptide bond formation are shown in stick format in yellow and the rest of SnoopTag colored magenta. (B) Sample gel to test for quantitative reaction of SnoopTag. Shown are 10 µM SnoopTag-MBP and 20 µM SnoopCatcher alone or mixed for 32 min (triplicate samples) before SDS/PAGE with Coomassie staining. (C) Sample gel to test for quantitative reaction of SnoopCatcher. Shown are 10 µM SnoopCatcher and 40 µM SnoopTag-MBP alone or mixed for the indicated times (triplicate samples) before SDS/PAGE with Coomassie staining. (D) Buffer dependence of SnoopTag/SnoopCatcher reaction. We incubated 10 µM SnoopTag-MBP with 10 µM SnoopCatcher at pH 8.0 for 15 min at 25 °C in the indicated buffer and analyzed the samples by SDS/PAGE with Coomassie staining. (E) TMAO dependence of SnoopTag/SnoopCatcher reaction tested as in D. Error bars are all mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Incubation