Recombinant Production of E. coli NAD+-dependent DNA ligase as a Target for Antibacterial Drug Discovery
Öz
The increase in the frequency of drug resistance in bacterial infections has led to the research of new antibacterial agents targeting new mechanisms. Many of the functions of NAD+-dependent DNA ligase have made it a remarkable target for antibacterial drug discovery. Escherichia coli (E. coli) NAD+-dependent DNA ligase is presented as a potential target due to its unique substrate specificity compared to the ATP-dependent human DNA ligase. In this study, it was aimed to produce and purify the E. coli NAD + dependent DNA ligase enzyme, which is frequently used in antibacterial drug discovery. The E. coli DNA ligase gene sequence was cloned into pTOLT vector system. E. coli DNA ligase enzyme was purified after the production in E. coli BL21 (DE3) pLysE cells. It was clearly demonstrated by the activity test that the DNA ligase enzyme produced in this study can ligate the DNA fragments. As a result, it was revealed that the effect of candidate inhibitors can be studied simply on the enzyme.
Anahtar Kelimeler
NAD+-dependent DNA ligase TolAIII peptide Antibacterial Drug Discovery
Destekleyen Kurum
Turkish Scientific and Technical Research Council (TUBITAK)
Proje Numarası
2209-A
Teşekkür
This study was supported by the Turkish Scientific and Technical Research Council (TUBITAK) (TUBITAK-2209A)
Kaynakça
- Alomari A, Allen, M 2019. Clone, purify and 1D Nuclear Magnetic Resonance Spectroscopy of the BRCT Domain of E. coli DNA Ligase LigA. Iraqi Journal of Biotechnologies 18, 2.
- Anderluh G, Gokce I, Lakey JH 2003. Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner. Protein Expr Purif 28:173-181. doi:10.1016/s1046-5928(02)00681-2.
- Brotz-Oesterhelt H, Knezevic I, Bartel S, Lampe T, Warnecke-Eberz U, Ziegelbauer K, Habich D, Labischinski H 2003. Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones. J Biol Chem 278:39435-39442. doi:10.1074/jbc.M306479200.
- Cardona-Felix CS, Pastor-Palacios G, Cardenas H, Azuara-Liceaga E, Brieba LG 2010. Biochemical characterization of the DNA ligase I from Entamoeba histolytica. Mol Biochem Parasitol 174:26-35. doi:10.1016/j.molbiopara.2010.06.010.
- Crut A, Nair PA, Koster DA, Shuman S, Dekker NH 2008. Dynamics of phosphodiester synthesis by DNA ligase. Proc Natl Acad Sci U S A 105:6894-6899. doi:10.1073/pnas.0800113105.
- Dermody JJ, Robinson GT, Sternglanz R 1979. Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli. J Bacteriol 139:701-704.
- Doherty AJ, Ashford SR, Wigley DB 1996. Characterization of proteolytic fragments of bacteriophage T7 DNA ligase. Nucleic Acids Res 24:2281-2287. doi:10.1093/nar/24.12.2281.
- Dwivedi N, Dube D, Pandey J, Singh B, Kukshal V, Ramachandran R, Tripathi RP (2008) NAD(+)-dependent DNA ligase: a novel target waiting for the right inhibitor. Med Res Rev 28:545-568. doi:10.1002/med.20114.
- Georlette D, Blaise V, Dohmen C, Bouillenne F, Damien B, Depiereux E., Gerday C, Uversky VN, Feller G 2003. Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus. J Biol Chem 278:49945-49953. doi:10.1074/jbc.M307761200.
- Gong C, Martins A, Bongiorno P, Glickman M, Shuman S 2004. Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem 279:20594-20606. doi:10.1074/jbc.M401841200.
- Jonsson ZO, Thorbjarnardottir SH, Eggertsson G, Palsdottir A 1994. Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases. Gene 151:177-180. doi:10.1016/0378-1119(94)90652-1.
- Kaczmarek FS, Zaniewski RP, Gootz TD, Danley DE, Mansour MN, Griffor M, Kamath AV, Cronan M, Mueller J, Sun D, Martin PK, Benton B, McDowell L, Biek D, Schmid MB 2001. Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus. J Bacteriol 183:3016-3024. doi:10.1128/JB.183.10.3016-3024.2001.
- Kaplan Ö, İmamoğlu R, Şahingöz İ, Gökçe İ 2021. Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer. International Advanced Researches and Engineering Journal. 5(1): 42-46. doi:10.35860/iarej.766741.
- Korycka-Machala M, Rychta E, Brzostek A, Sayer HR, Rumijowska-Galewicz A, Bowater RP, Dziadek J 2007. Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics. Antimicrob Agents Chemother 51:2888-2897. doi:10.1128/AAC.00254-07.
- Kuduğ H, Ataman B, İmamoğlu R, Düzgün D, Gökçe İ 2019. Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. International Advanced Researches and Engineering Journal. 3(1): 20-25.
- Lauer G, Rudd EA, McKay DL, Ally A, Ally D, Backman KC 1991. Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase. J Bacteriol 173:5047-5053. doi:10.1128/jb.173.16.5047-5053.1991.
- Lee JY, Chang C, Song HK, Moon J, Yang JK, Kim HK, Kwon ST, Suh SW 2000. Crystal structure of NAD(+)- dependent DNA ligase: modular architecture and functional implications. EMBO J 19:1119-1129. doi:10.1093/emboj/19.5.1119.
- Meier TI, Yan D, Peery RB, McAllister KA, Zook C, Peng SB, Zhao G 2008. Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases. FEBS J 275:5258-5271. doi:10.1111/j.1742-4658.2008.06652.x.
- Mills SD, Eakin AE, Buurman ET, Newman JV, Gao N, Huynh H, Johnson KD, Lahiri S, Shapiro AB, Walkup GK, Yang W, Stokes SS 2011. Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo. Antimicrob Agents Chemother 55:1088-1096. doi:10.1128/AAC.01181-10.
- Odell M, Sriskanda V, Shuman S, Nikolov DB 2000. Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining. Mol Cell 6:1183-1193. doi:10.1016/s1097-2765(00)00115-5.
- Seo MS, Kim YJ, Choi JJ, Lee MS, Kim JH, Lee JH, Kwon ST 2007. Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme. J Biotechnol 128:519-530. doi:10.1016/j.jbiotec.2006.09.024.
- Shuman S 2009. DNA ligases: progress and prospects. J Biol Chem 284:17365-17369. doi:10.1074/jbc.R900017200.
- Sriskanda V, Shuman S 2002. Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+. J Biol Chem 277:9695-9700. doi:10.1074/jbc.M111164200.
- Srivastava SK, Dube D, Kukshal V, Jha AK, Hajela K, Ramachandran R 2007. NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor. Proteins 69:97-111. doi:10.1002/prot.21457.
- Subramanya HS, Doherty AJ, Ashford SR, Wigley DB 1996. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell 85:607-615. doi:10.1016/s0092-8674(00)81260-x.
- Swift RV, Amaro RE 2009. Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms. Expert Opin Drug Discov 4:1281-1294. doi:10.1517/17460440903373617.
- Timson DJ, Singleton MR, Wigley DB 2000. DNA ligases in the repair and replication of DNA. Mutat Res 460:301-318. doi:10.1016/s0921-8777(00)00033-1.
- Wilkinson A, Day J, Bowater R 2001. Bacterial DNA ligases. Mol Microbiol 40:1241-1248. doi:10.1046/j.1365-2958.2001.02479.x.
- Wilkinson A, Smith A, Bullard D, Lavesa-Curto M, Sayer H, Bonner A, Hemmings A, Bowater R 2005. Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA). Biochim Biophys Acta 1749:113-122. doi:10.1016/j.bbapap. 2005.03.003.
Antibakteriyel İlaç Keşfi İçin Bir Hedef Olarak E. Coli NAD+-Bağımlı DNA Ligazın Rekombinant Üretimi
Öz
Bakteriyel enfeksiyonlarda ilaç direnci frekansındaki artış, yeni mekanizmaları hedefleyen yeni antibakteriyel ajanların araştırılmasına yol açmıştır. NAD+-bağımlı DNA ligazın fonksiyonlarının bir çoğu, onu antibakteriyel ilaç keşfi için dikkate değer bir hedef haline getirmiştir. Escherichia coli (E. coli) NAD+ bağımlı DNA ligazı, ATP bağımlı insan DNA ligazı ile karşılaştrıldığında, benzersiz substrat özgüllüğü nedeniyle potansiyel bir hedef olarak görülmektedir. Bu çalışmada antibakteriyel ilaç keşiflerinde sıklıkla kullanılan E. coli NAD+ bağımlı DNA ligaz enziminin, yüksek miktarda ve saflıkta üretilmesi amaçlamıştır. E. coli DNA ligaz gen dizisi, pTOLT vektör sistemine klonlanmıştır. E. coli DNA ligaz enzimi, E. coli BL21 (DE3) pLysE hücrelerinde üretildikten sonra saflaştırılmıştır. Bu çalışmada üretilen DNA ligaz enziminin DNA fragmanlarının ligasyonunu sağlayabildiği, aktivite testi ile açıkça gösterilmiştir. Sonuç olarak, bu enzim üzerinde aday inhibitörlerin etkisinin basitçe incelenebileceği ortaya koyulmuştur.
Anahtar Kelimeler
NAD+-bağımlı DNA ligaz TolAIII peptid Antibakteriyel İlaç Keşfi
Proje Numarası
2209-A
Kaynakça
- Alomari A, Allen, M 2019. Clone, purify and 1D Nuclear Magnetic Resonance Spectroscopy of the BRCT Domain of E. coli DNA Ligase LigA. Iraqi Journal of Biotechnologies 18, 2.
- Anderluh G, Gokce I, Lakey JH 2003. Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner. Protein Expr Purif 28:173-181. doi:10.1016/s1046-5928(02)00681-2.
- Brotz-Oesterhelt H, Knezevic I, Bartel S, Lampe T, Warnecke-Eberz U, Ziegelbauer K, Habich D, Labischinski H 2003. Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones. J Biol Chem 278:39435-39442. doi:10.1074/jbc.M306479200.
- Cardona-Felix CS, Pastor-Palacios G, Cardenas H, Azuara-Liceaga E, Brieba LG 2010. Biochemical characterization of the DNA ligase I from Entamoeba histolytica. Mol Biochem Parasitol 174:26-35. doi:10.1016/j.molbiopara.2010.06.010.
- Crut A, Nair PA, Koster DA, Shuman S, Dekker NH 2008. Dynamics of phosphodiester synthesis by DNA ligase. Proc Natl Acad Sci U S A 105:6894-6899. doi:10.1073/pnas.0800113105.
- Dermody JJ, Robinson GT, Sternglanz R 1979. Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli. J Bacteriol 139:701-704.
- Doherty AJ, Ashford SR, Wigley DB 1996. Characterization of proteolytic fragments of bacteriophage T7 DNA ligase. Nucleic Acids Res 24:2281-2287. doi:10.1093/nar/24.12.2281.
- Dwivedi N, Dube D, Pandey J, Singh B, Kukshal V, Ramachandran R, Tripathi RP (2008) NAD(+)-dependent DNA ligase: a novel target waiting for the right inhibitor. Med Res Rev 28:545-568. doi:10.1002/med.20114.
- Georlette D, Blaise V, Dohmen C, Bouillenne F, Damien B, Depiereux E., Gerday C, Uversky VN, Feller G 2003. Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus. J Biol Chem 278:49945-49953. doi:10.1074/jbc.M307761200.
- Gong C, Martins A, Bongiorno P, Glickman M, Shuman S 2004. Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem 279:20594-20606. doi:10.1074/jbc.M401841200.
- Jonsson ZO, Thorbjarnardottir SH, Eggertsson G, Palsdottir A 1994. Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases. Gene 151:177-180. doi:10.1016/0378-1119(94)90652-1.
- Kaczmarek FS, Zaniewski RP, Gootz TD, Danley DE, Mansour MN, Griffor M, Kamath AV, Cronan M, Mueller J, Sun D, Martin PK, Benton B, McDowell L, Biek D, Schmid MB 2001. Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus. J Bacteriol 183:3016-3024. doi:10.1128/JB.183.10.3016-3024.2001.
- Kaplan Ö, İmamoğlu R, Şahingöz İ, Gökçe İ 2021. Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer. International Advanced Researches and Engineering Journal. 5(1): 42-46. doi:10.35860/iarej.766741.
- Korycka-Machala M, Rychta E, Brzostek A, Sayer HR, Rumijowska-Galewicz A, Bowater RP, Dziadek J 2007. Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics. Antimicrob Agents Chemother 51:2888-2897. doi:10.1128/AAC.00254-07.
- Kuduğ H, Ataman B, İmamoğlu R, Düzgün D, Gökçe İ 2019. Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. International Advanced Researches and Engineering Journal. 3(1): 20-25.
- Lauer G, Rudd EA, McKay DL, Ally A, Ally D, Backman KC 1991. Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase. J Bacteriol 173:5047-5053. doi:10.1128/jb.173.16.5047-5053.1991.
- Lee JY, Chang C, Song HK, Moon J, Yang JK, Kim HK, Kwon ST, Suh SW 2000. Crystal structure of NAD(+)- dependent DNA ligase: modular architecture and functional implications. EMBO J 19:1119-1129. doi:10.1093/emboj/19.5.1119.
- Meier TI, Yan D, Peery RB, McAllister KA, Zook C, Peng SB, Zhao G 2008. Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases. FEBS J 275:5258-5271. doi:10.1111/j.1742-4658.2008.06652.x.
- Mills SD, Eakin AE, Buurman ET, Newman JV, Gao N, Huynh H, Johnson KD, Lahiri S, Shapiro AB, Walkup GK, Yang W, Stokes SS 2011. Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo. Antimicrob Agents Chemother 55:1088-1096. doi:10.1128/AAC.01181-10.
- Odell M, Sriskanda V, Shuman S, Nikolov DB 2000. Crystal structure of eukaryotic DNA ligase-adenylate illuminates the mechanism of nick sensing and strand joining. Mol Cell 6:1183-1193. doi:10.1016/s1097-2765(00)00115-5.
- Seo MS, Kim YJ, Choi JJ, Lee MS, Kim JH, Lee JH, Kwon ST 2007. Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme. J Biotechnol 128:519-530. doi:10.1016/j.jbiotec.2006.09.024.
- Shuman S 2009. DNA ligases: progress and prospects. J Biol Chem 284:17365-17369. doi:10.1074/jbc.R900017200.
- Sriskanda V, Shuman S 2002. Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+. J Biol Chem 277:9695-9700. doi:10.1074/jbc.M111164200.
- Srivastava SK, Dube D, Kukshal V, Jha AK, Hajela K, Ramachandran R 2007. NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor. Proteins 69:97-111. doi:10.1002/prot.21457.
- Subramanya HS, Doherty AJ, Ashford SR, Wigley DB 1996. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell 85:607-615. doi:10.1016/s0092-8674(00)81260-x.
- Swift RV, Amaro RE 2009. Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms. Expert Opin Drug Discov 4:1281-1294. doi:10.1517/17460440903373617.
- Timson DJ, Singleton MR, Wigley DB 2000. DNA ligases in the repair and replication of DNA. Mutat Res 460:301-318. doi:10.1016/s0921-8777(00)00033-1.
- Wilkinson A, Day J, Bowater R 2001. Bacterial DNA ligases. Mol Microbiol 40:1241-1248. doi:10.1046/j.1365-2958.2001.02479.x.
- Wilkinson A, Smith A, Bullard D, Lavesa-Curto M, Sayer H, Bonner A, Hemmings A, Bowater R 2005. Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA). Biochim Biophys Acta 1749:113-122. doi:10.1016/j.bbapap. 2005.03.003.
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Bölüm | ARAŞTIRMA MAKALESİ (Research Article) |
| Yazarlar | |
| Proje Numarası | 2209-A |
| Yayımlanma Tarihi | 28 Şubat 2022 |
| Gönderilme Tarihi | 21 Şubat 2021 |
| Kabul Tarihi | 20 Nisan 2021 |
| Yayımlandığı Sayı | Yıl 2022Cilt: 25 Sayı: 1 |
2022-JIF = 0.500
2022-JCI = 0.170
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