The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis
Abstract
:1. Introduction
2. Results
2.1. The Detection of Soluble Tb-DdlA Protein Expressed in Escherichia coli
2.2. The Profiles of Tb-DdlA d-Alanyl-d-alanine Ligase Activity
2.2.1. Confirmation of DdlA Activity
2.2.2. Kinetic Analysis of Tb-DdlA
2.2.3. Inhibitory Analysis of Tb-DdlA Using DCS
2.3. The Identification of Tb-DdlA Potential Interaction Partners
3. Discussion
4. Methods
4.1. Strains, Plasmids, and Growth Conditions
4.2. Construction of Tb-ddlA Expression Plasmids
4.3. Expression, Purification, and Identification of Tb-DdlA
4.4. Functional Assay of Tb-DdlA
4.5. Kinetic Analysis of Tb-DdlA
4.6. Colorimetric Inhibition Assay of Tb-DdlA by DCS
4.7. Pull down Assay of Tb-DdlA
4.8. MS/MS Analysis
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sample Availability: Samples of the pCold II-Tb-ddlAplasmid are available from the authors. |
Reactions | Components | Mean Values of A620nm ± SD | Net Mean ± SD | Z-Factor | |
---|---|---|---|---|---|
Reaction 1 | 100 μmol/L ATP; 2 mmol/L d-Ala | 20 μg/mL DdlA | 0.692 ± 0.099 | 0.452 ± 0.099 | 0.187 |
100 μmol/L ATP; 2 mmol/L d-Ala | No DdlA | 0.240 ± 0.023 | |||
Reaction 2 | 75 μmol/L ATP; 2 mmol/L d-Ala | 10 μg/mL DdlA | 0.459 ± 0.013 | 0.291 ± 0.013 | 0.735 |
75 μmol/L ATP; 2 mmol/L d-Ala | No DdlA | 0.168 ± 0.013 |
ID | Protein Name | Accession No. (NCBI) | Locus Name/Gene Definition | Protein MW (D) | Protein PI | Pep. Count | Protein Score | Protein Score C. I. % |
---|---|---|---|---|---|---|---|---|
A | LuxR family transcriptional regulator | AIU17347.1 | MSMEG_5707, cupin | 12,011.3 | 6.36 | 5 | 285 | 100 |
B | hypothetical protein LI98_12890 | AIU21016.1 | MSMEG_2589 | 15,412.9 | 6.28 | 7 | 314 | 100 |
C | elongation factor Tu | YP_885786.1 | MSMEG_1401, tuf | 43,708.6 | 5.18 | 18 | 696 | 100 |
FAD-dependent oxidoreductase | AIU20161.1 | MSMEG_1682, FMO | 46,185.2 | 5.86 | 13 | 74 | 99.697 | |
ornithine-oxo-acid transaminase | WP_011727654.1 | MSMEG_1413, rocD | 44,661.1 | 5.62 | 14 | 72 | 99.581 | |
D | diaminopimelate decarboxylase | YP_889210.1 | MSMEG_4958, lysA | 50,317 | 5.25 | 13 | 224 | 100 |
cyclopropane-fatty-acyl-phospholipid synthase | YP_890503.1 | MSMEG_6284 | 48,608.7 | 5.99 | 13 | 198 | 100 | |
carbon-monoxide dehydrogenase large subunit | WP_011727162.1 | MSMEG_0746 | 85,807 | 5.21 | 19 | 60 | 93.366 |
Strains/Plasmids | Description | Source |
---|---|---|
Strains | ||
E. coli NovaBlue | Used for cloning and propagation of plasmids | Novagen |
E. coli BL21(DE3) | Used for expressing Tb-DdlA protein | Invitrogen |
Plasmids | ||
pMD18-T | Carries ampR gene; used for cloning PCR product with A at 3′ ends | Takara |
pCold II | Carries ampR gene; contains cold start promoter; used for expressing M. tuberculosis DdlA | Takara |
pMD18-Tb-ddlA | Carries ampR gene; M. tuberculosis ddlA was cloned to the EcoRV site of pMD18-T | This study |
pCold II-Tb-ddlA | Carries ampR gene; used for expressing M. tuberculosis DdlA | This study |
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Yang, S.; Xu, Y.; Wang, Y.; Ren, F.; Li, S.; Ding, W.; Ma, Y.; Zhang, W. The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis. Molecules 2018, 23, 324. https://doi.org/10.3390/molecules23020324
Yang S, Xu Y, Wang Y, Ren F, Li S, Ding W, Ma Y, Zhang W. The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis. Molecules. 2018; 23(2):324. https://doi.org/10.3390/molecules23020324
Chicago/Turabian StyleYang, Shufeng, Yuefei Xu, Yan Wang, Feng Ren, Sheng Li, Wenyong Ding, Yufang Ma, and Wenli Zhang. 2018. "The Biological Properties and Potential Interacting Proteins of d-Alanyl-d-alanine Ligase A from Mycobacterium tuberculosis" Molecules 23, no. 2: 324. https://doi.org/10.3390/molecules23020324