Recombinant Production of E. coli NAD+-dependent DNA ligase as a Target for Antibacterial Drug Discovery

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. Research Article Article History Geliş Tarihi : 21.02.2021 Kabul Tarihi : 20.04.2021


INTRODUCTION
DNA ligase is important in DNA repair and replication (Cardona-Felix et al. 2010). DNA synthesis in one strand may be continuous in DNA replication, while synthesis is not continuous in the other strand. DNA ligase is crucial for joining Okazaki fragments and fulfilling DNA synthesis (Timson et al. 2000). Nicks caused by environmental factors such as radiation, oxidants and alkylating agents which are repaired by the activity of the DNA ligase (Crut et al. 2008). DNA ligase is an essential protector of genome integrity (Odell et al. 2000). Various organisms such as E. coli (Wilkinson et al. 2005), Mycobacterium tuberculosis (M. tuberculosis) (Srivastava et al. 2007  , Staphylothermus marinus (S. marinus) (Seo et al. 2007) have ATPdependent DNA ligase. In addition, all known eukaryotic DNA ligases are ATP dependent (Wilkinson et al. 2001). Bacterial genomes sequenced so far contain at least one NAD + -dependent DNA ligase. NAD + -dependent ligase has been cloned from various bacterial sources and sequenced. These sequences appear to be significantly similar and all have almost the same size (75 kDa) (Timson et al. 2000). These enzymes have two functional domains: the C terminal domain is the domain of binding to DNA. It is seen that a zinc ion is bound in the C terminal domain of these enzymes. The N terminal domain is responsible for the self-adenylating reaction and NAD + binding (Wilkinson et al. 2001). The DNA sequence of the DNA ligase gene of a wide range of bacterial species (Jonsson et al. 1994;Lauer et al. 1991;Subramanya et al. 1996;Timson et al. 2000) has been identified, and some conserved regions have been determined and compared with the discovery regions in E. coli. Alignment of amino acid sequences displays that S. aureus NAD + -dependent DNA ligase has 47% and 60% amino acid identity with E. coli and Bacillus stearothermophilus NAD + -dependent DNA ligase, respectively. It has been displayed in further studies that these DNA ligases have motifs preserved in other bacteria (Kaczmarek et al. 2001). E. coli DNA is a protein encoded by the LigA gene, the enzyme has 671 amino acids and its molecular weight is 73,690 kDa. Wilkinson et al. (2005) found that E. coli LigA protein has contains zinc-finger, oligomerbinding (OB) h-barrel, helix-hairpin-helix motif (HhH) and BRCT domain. In the same study, E. coli LigA gene was cloned into the pET16b vector. The vector contains a 10-His-Tag containing 21 extra amino acids at the N-terminal of protein. Also, it was stated that the presence of His-Tag at the N-terminal or C-terminal end did not affect enzyme activity (Wilkinson et al. 2005).
NAD + -dependent DNA ligases are considered to be possible targets for broad spectrum antibacterial compounds (Gong et al. 2004;Kaczmarek et al. 2001;Lee et al. 2000;Sriskanda and Shuman 2002;Swift and Amaro 2009), because they are unavoidable for many basic processes of DNA metabolism, including replication, recombination processes and repairs that require re-synthesis of DNA connection of Okazaki fragments in the pathway (Timson et al. 2000). The ligase inhibitors developed are generally competitive inhibitors and can bind to hydrophobic pockets near the NAD + substrate binding site (Swift and Amaro 2009  In this study, E. coli NAD+-dependent DNA ligase enzyme, which is frequently used in antibacterial drug discoveries, was aimed at high level production and purification. First, the E. coli DNA ligase gene was cloned into the pTOLT vector system and then expressed in E. coli BL21 (DE3) pLysE cells. The enzyme was purified by affinity chromatography and its activity was demonstrated.

MATERIALS and METHOD
Cloning of the E. coli DNA Ligase Gene into the pTOLT

Vector System
The gene was first amplified for cloning the E. coli DNA Ligase gene into the pTOLT vector (Anderluh et al. 2003). PCR (polymerase chain reaction) mixture was prepared using primers of ligABamHIsense 5'TTTTGGATCCATGGAATCAATCGAACAAC3' and ligAKpnIreverse 5'TTTTGGTACCGCTACCCAGCAA ACGCAG3'.
After PCR reaction, 10 µl of PCR mixture was taken and analyzed in 1% agarose gel. The obtained PCR products were purified with the PCR products cleaning kit and digested with BamHI and KpnI restriction enzymes. The pTOLT plasmid which was used for cloning was also cut with the same restriction enzymes. E. coli DNA Ligase gene and pTOLT plasmid digestion by restriction enzymes were ligated with T4 DNA ligase enzyme at room temperature for 16 hours.
The ligation products were transferred to E. coli DH5α cells and spread on ampicillin (100 µg ml -1 ) containing LB medium. Plasmid DNA was isolated from the colonies, and diagnostic restriction digest and diagnostic PCR were performed on plasmids. The obtained products were analyzed in 1% agarose gel.
Expression of E. coli DNA Ligase Protein Protein expression and purification was carried out similar to the protocol detailed in our previous work (Kuduğ et al. 2019;Kaplan et al. 2021). In summary, for protein expression, pTOLT clones were transformed into E. coli BL21 (DE3) pLysE and spread on LB medium containing ampicillin (100 µg ml -1 ). KSU J. Agric Nat 25 (1) As a result of diagnostic PCR and diagnostic restriction analysis, E. coli LigA gene has been successfully cloned into pTOLT vector and, the pTOL-E. coli DNA Ligase plasmid was obtained (Figure 4).

Expression of E. coli DNA Ligase Protein
The pTOLT plasmid has a DNA sequence encoding the TolAIII peptide of 9 kDa as fusion. TolAIII is a small domain, expressed in high yields of soluble proteins in the cytoplasm of E. coli. The E. coli DNA ligase enzyme is 74 kDa in size and appears to be 83 kDa with the 9 kDa TolAIII peptide in SDS-PAGE gel ( Figure 5). KSU J. Agric Nat 25 (1) (Dermody et al. 1979;Kaczmarek et al. 2001;Meier et al. 2008;Shuman 2009). NAD + -dependent DNA ligases were not detected in humans, which led to the research that it could be the target for broad-spectrum antibiotics (Brotz-Oesterhelt et al. 2003;Georlette et al. 2003;Gong et al. 2004;Kaczmarek et al. 2001;Lee et al. 2000;Sriskanda and Shuman (2002) spectrum antibiotics (Brotz-Oesterhelt et al. 2003;Georlette et al. 2003;Gong et al. 2004;Kaczmarek et al. 2001;Lee et al. 2000;Sriskanda and Shuman 2002). NAD + -dependent DNA Ligases have low similarity with eukaryotic DNA ligase, and the need for NAD + as a substrate for ligase activity makes the bacterial ligase unique, thus limiting toxicity for humans (Korycka-Machala et al. 2007). Ligases are found in all three systems of life (bacteria, archaea and eukaryotes), while NAD + -dependent enzymes are only present in bacteria (Wilkinson et al. 2001). At least one NAD + -dependent DNA ligase is found in each bacterium. Genetic studies have demonstrated that the LigA gene is required for E. coli growth (Shuman 2009). NAD + -dependent ligase (LigA) is an essential enzyme for the survival of E. coli and its homologous protein may be required for all bacteria (Wilkinson et al. 2001).
In this study, the E. coli DNA ligase gene fragment was cloned into the pTOLT vector system. The protein expression was performed in E. coli BL21 (DE3) pLysE cells and the protein was purified. The E. coli DNA ligase protein was highly expressed by fusion with the TolAIII peptide. The greatest advantage of expression in combination with TolAIII is that it provides high efficiency protein production in E. coli (Anderluh et al. 2003). The TolAIII peptide significantly increased the production of E. coli DNA ligase protein. The activity of the purified protein was confirmed by an activity test. It has been revealed that the effect of selected inhibitors can be studied simply on the enzyme we produce within the scope of this study. In addition, the produced E. coli DNA ligase enzyme can also be used in molecular cloning processes.

ACKNOWLEDGEMENT
This study was supported by the Turkish Scientific and Technical Research Council (TUBITAK) (TUBITAK-2209A)

Researchers Contribution Rate Declaration Summary
The authors declare that they have contributed equally to the article.