Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (XY33a_V-4K_gene) |
XY33a_V-4K_gene | IDT. Representative of other purchased XY33a mutant genes (see Table 5) |
XY33a_V-4K_gene | 5 ng/μL stock solution (1 μL) used as amplification template |
| Strain, strain background (Escherichia coli BL21 (DE3)-T1R) |
E. coli BL21 (DE3)-T1R | Sigma Aldrich B2935 | BL21 (DE3)-T1R | |
| Strain, strain background (Lachnospiraceae C6A11) |
Lachnospiraceae C6A11 | Dr. William Kelly (AgResearch, New Zealand) |
Lachnospiraceae C6A11 | |
| Strain, strain background (Escherichia coli Rosetta 2 (DE3)) |
E. coli Rosetta 2 (DE3) | Novagen Catalog no. 71400–3 | E. coli Rosetta 2 (DE3) | |
| Strain, strain background (Azospirillum sp. B510 (JCM 14679)) |
Azospirillum sp. B510 (JCM 14679) |
JCM Riken http://www.jcm.riken.jp/cgi-bin/jcm/jcm_number?JCM=14679 |
Azospirillum sp. B510 (JCM 14679) | |
| Transformed construct (pETDuet LahT150) |
pETDuet-LahT150 | this work | pETDuet LahT150 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet XY33a) |
pRSFDuet-XY33a | PMID: 22574919 | pRSFDuet XY33a | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet XY33a A-3Y) |
pRSFDuet XY33a A-3Y | this work. Representative XY33a mutant |
pRSFDuet XY33a A-3Y | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA1) |
pRSFDuet-LahA1 | this work | pRSFDuet LahA1 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA2) |
pRSFDuet-LahA2 | this work | pRSFDuet LahA2 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA3) |
pRSFDuet-LahA3 | this work | pRSFDuet LahA3 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA4) |
pRSFDuet-LahA4 | this work | pRSFDuet LahA4 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA5) |
pRSFDuet-LahA5 | this work | pRSFDuet LahA5 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA6) | pRSFDuet-LahA6 | this work | pRSFDuet LahA6 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet LahA7) | pRSFDuet-LahA7 | this work | pRSFDuet LahA7 | 50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pET28-MBP-AzoA2) |
pET28-MBP-AzoA2 | this work | pET28-MBP-AzoA2 | 150 ng/μL stock solution (1 μL) used in E. coli Rosetta 2 (DE3) transformation |
| Transformed construct (pET28-MBP-AzoA3) | pET28-MBP-AzoA3 | this work | pET28-MBP-AzoA3 | 150 ng/μL stock solution (1 μL) used in E. coli Rosetta 2 (DE3) transformation |
| Transformed construct (pET28-MBP-AzoA6) |
pET28-MBP-AzoA6 | this work | pET28-MBP-AzoA6 | 150 ng/μL stock solution (1 μL) used in E. coli Rosetta 2 (DE3) transformation |
| Transformed construct (pET28-MBP-AzoA7) | pET28-MBP-AzoA7 | this work | pET28-MBP-AzoA7 | 150 ng/μL stock solution (1 μL) used in E. coli Rosetta 2 (DE3) transformation |
| Transformed construct (pRSFDuet ProcA 2.8 (MCSI) - ProcM (MCSII)) |
pRSFDuet ProcA2.8 (MCSI) - ProcM (MCSII) |
PMID: 22574919 | pRSFDuet ProcA 2.8 (MCSI) - ProcM (MCSII) |
50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet ProcA 1.7 (MCSI) - ProcM (MCSII)) |
pRSFDuet ProcA1.7 (MCSI) - ProcM (MCSII |
PMID: 22574919 | pRSFDuet ProcA 1.7 (MCSI) - ProcM (MCSII |
50 ng/μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet ProcA 2.1 (MCSI) - ProcM (MCSII)) |
pRSFDuet ProcA2.1 (MCSI) - ProcM (MCSII |
this work | pRSFDuet ProcA 2.1 (MCSI) - ProcM (MCSII |
50 ng/ μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet ProcA 2.4 (MCSI) - ProcM (MCSII)) |
pRSFDuet ProcA2.4 (MCSI) - ProcM (MCSII) |
this work | pRSFDuet ProcA 2.4 (MCSI) - ProcM (MCSII) |
50 ng/ μL stock solution (1 μL) used in E. coli BL21 transformation |
| Transformed construct (pRSFDuet ProcA 1.3 (MCSI) - ProcM (MCSII)) |
pRSFDuet ProcA1.3 (MCSI) - ProcM (MCSII) |
this work | pRSFDuet ProcA 1.3 (MCSI) - ProcM (MCSII) |
50 ng/ μL stock solution (1 μL) used in E. coli BL21 transformation |
| Sequence- based reagent |
Benzonase Endonuclease |
EMD Millipore Catalog no. 1.01656.001 |
Benzonase | |
| Sequence- based reagent |
EcoRI-HF | New England Biolabs R3101S |
EcoRI | |
| Sequence- based reagent |
BamHI-HF | New England Biolabs R3136S |
BamHI | |
| Sequence- based reagent |
NotI-HF | New England Biolabs R3189S |
Not1 | |
| Sequence- based reagent |
HindIII-HF | New England Biolabs R3104S |
HindIII | |
| Recombinant protein |
XY33a | PMID: 29507389 | XY33a | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
XY33a A-3Y | this work; representative XY33a mutant |
XY33a A-3Y | recombinant substrate peptide mutant tested with LahT150 |
| Recombinant protein |
LahA1 | this work | LahA1 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahA2 | this work | LahA2 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahA3 | this work | LahA3 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahA4 | this work | LahA4 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahA5 | this work | LahA5 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahA6 | this work | LahA6 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahA7 | this work | LahA7 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
AzoA2 | this work | MBP-AzoA2 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
AzoA3 | this work | MBP-AzoA3 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
AzoA6 | this work | MBP-AzoA6 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
AzoA7 | this work | MBP-AzoA7 | recombinant substrate peptide tested with LahT150 |
| Recombinant protein |
LahT150 | this work | LahT150 | protease domain of LahT |
| Recombinant protein |
Pcn 2.8 | PMID: 22574919 | Pcn 2.8 | recombinant posttranslationally modified ProcA2.8 substrate peptide tested with LahT150 |
| Recombinant protein |
Pcn 1.7 | PMID: 22574919 | Pcn 1.7 | recombinant posttranslationally modified ProcA1.7 substrate peptide tested with LahT150 |
| Recombinant protein |
Pcn 2.1 | this work | Pcn 2.1 | recombinant posttranslationally modified ProcA2.1 substrate peptide tested with LahT150 |
| Recombinant protein |
Pcn 2.4 | this work | Pcn 2.4 | recombinant posttranslationally modified ProcA2.4 substrate peptide tested with LahT150 |
| Recombinant protein | Pcn 1.3 | this work | Pcn 1.3 | recombinant posttranslationally modified ProcA1.3 substrate peptide tested with LahT150 |
| Minimum peptide substrate | Synthetic peptide | Genscript | Synthetic peptide | synthetic minimal substrate peptide tested with LahT150 |
| Commercial kit |
QIAprep Spin Miniprep kit |
Qiagen catalog no. 27106 |
QIAprep Spin Miniprep kit |
|
| Commercial kit |
QIAquick Gel Extraction kit |
Qiagen catalog no. 28115 |
QIAquick Gel Extraction kit |
|
| Commercial kit |
Gibson Assembly | New England Biolabs E2611S |
Gibson Assembly | |
| Chemical compound |
TCEP (Tris (2-Carboxyethyl) phosphine hydrochloride) |
Goldbio Catalog ID TCEP |
TCEP | |
| Chemical compound |
Terrific Broth granulated |
Fisher Scientific BP97285 |
TB | |
| Chemical compound |
Glycerol | Fisher Scientific BP-229–4 |
Glycerol | |
| Chemical compound |
Dextrose | Fisher Scientific BP350500 |
Glucose or dextrose | |
| Chemical compound |
kanamycin monosulfate, USP grade |
Goldbio Catalog ID K-120 |
kanamycin | |
| Software, algorithm |
Adobe Illustrator CS6 | Adobe | Adobe Illustrator | |
| Software, algorithm |
FlexAnalysis 3.4 (Bruker Daltonik GmbH) |
Bruker Daltonik GmbH |
FlexAnalysis 3.4 (Bruker Daltonik GmbH) |
Mass spectrometry data processing |
| Other | Clontech His60 Ni Superflow resin |
Clontech Catalog no. 636660 |
Clontech His60 Ni Superflow resin |
Used for gravity purification of all recombinant proteins except LahT150 |
| Other | GE Healthcare HisTrap HP |
GE Healthcare 17524701 | 5 mL HiTrap Ni Chelating column |
Used for FPLC purification of recombinant LahT150 |