Background: Reagent proteins such as DNA ligases play a central role in the global reagents market. DNA ligases are commonly used and are vital in academic and science research environments. Their major functions include sealing nicks by linking the 5'-phosphorylated end to a 3'-hydroxyl end on the phosphodiester backbone of DNA, utilizing ATP or NADP molecules as an energy source.
Objective: The current study sought to investigate the role of PEGylation on the biological activity of purified recombinant DNA ligases.
Methods: We produced two recombinant DNA ligases (Ligsv081 and LigpET30) using E. coli expression system and subsequently purified using affinity chromatography. The produced proteins wereconjugated to site specific PEGylation or non-specific PEGylation. FTIR and UV-VIS spectroscopy were used to analyze secondary structures of the PEG conjugated DNA ligases. Differential scanning fluorimetry was employed to assess the protein stability when subjected to various PEGylation conditions.
Results: In this study, both recombinant DNA ligases were successfully expressed and purified as homogenous proteins. Protein PEGylation enhanced ligation activity, increased transformation efficiency by 2-foldfor plasmid ligations and reduced the formation of protein aggregates.
Conclusion: Taken together, site-specific PEGylation can potentially be explored to enhance the biological activity and stability of reagent proteins such as ligases.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.2174/0929866529666220426122432 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Ring finger protein 5 mediates STING degradation through ubiquitinating K135 and K155 in a teleost fish.
Front Immunol
December 2024
School of Marine Sciences, State Key Laboratory for Biocontrol/Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering & Guangdong Provincial Observation and Research Station for Marine Ranching of the Lingdingyang Bay, Sun Yat-sen University, Guangzhou, China.
Stimulator of interferon genes (STING) is a key connector protein in interferon (IFN) signaling, crucial for IFN induction during the activation of antiviral innate immunity. In mammals, ring finger protein 5 (RNF5) functions as an E3 ubiquitin ligase, mediating STING regulation through K150 ubiquitylation to prevent excessive IFN production. However, the mechanisms underlying RNF5's regulation of STING in teleost fish remain unknown.
View Article and Find Full Text PDFDNA Ligase I Circularises Potato Spindle Tuber Viroid RNA in a Biomolecular Condensate.
Mol Plant Pathol
December 2024
Plant Molecular and Cell Biology Program, University of Florida, Gainesville, Florida, USA.
Viroids are single-stranded circular noncoding RNAs that mainly infect crops. Upon infection, nuclear-replicating viroids engage host DNA-dependent RNA polymerase II for RNA-templated transcription, which is facilitated by a host protein TFIIIA-7ZF. The sense-strand and minus-strand RNA intermediates are differentially localised to the nucleolus and nucleoplasm regions, respectively.
View Article and Find Full Text PDFCRL3 ubiquitin ligase and Integrator phosphatase form parallel mechanisms to control early stages of RNA Pol II transcription.
Mol Cell
December 2024
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. Electronic address:
Control of RNA polymerase II (RNA Pol II) through ubiquitylation is essential for the DNA-damage response. Here, we reveal a distinct ubiquitylation pathway in human cells, mediated by CRL3, that targets excessive and defective RNA Pol II molecules at the initial stages of the transcription cycle. Upon ARMC5 loss, RNA Pol II accumulates in the free pool and in the promoter-proximal zone but is not permitted into elongation.
View Article and Find Full Text PDFTrim13-induced ubiquitination of RPS27A inhibits the progression of lung cancer by depending on the inactivation of NF-κB signaling pathway.
Physiol Rep
December 2024
Division of Life Sciences and Medicine, Department of Respiratory Medicine, the First Affiliated Hospital of USTC, University of Science and Technology of China (Anhui Provincial Cancer Hospital), Hefei, Anhui, China.
Lung cancer (LC) is the leading cause of cancer-related death worldwide. Recent studies have shown that tripartite motif 13 (TRIM13) play important regulatory roles in the progression of different tumors. In this study, we focused on the role of TRIM13 in LC tumorigenesis and its underlying molecular mechanisms.
View Article and Find Full Text PDF[A case of neonatal Mitochondrial DNA depletion syndrome type 13 caused by FBXL4 gene mutation].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi
December 2024
Department of Neonatal Intensive Care Unit, National Clinical Research Center for Child Health, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310052, China.
Objective: To explore the clinical phenotypes and genetic variant in a neonatal case of Mitochondrial DNA depletion syndrome type 13 (MTDPS13).
Methods: Clinical data and results of genetic testing of a neonate admitted to the Children's Hospital of Zhejiang University School of Medicine in January 2023 was retrospectively analyzed. The study was approved by the Medical Ethics Committee of the Children's Hospital of Zhejiang University.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!