Recombinant Methanosarcina barkeri Pyrrolysine--tRNA ligase (pylS) is expressed in E.coli as a full-length protein comprising amino acids 1 to 419, with an N-terminal 6xHis tag for simplified purification. The protein reaches purity levels above 85% as determined by SDS-PAGE, which appears to ensure dependable performance in research applications. This product is suitable for research use only, not for therapeutic or diagnostic purposes.
Pyrrolysine--tRNA ligase, encoded by the pylS gene, plays a critical role in translation machinery by helping incorporate the rare amino acid pyrrolysine into proteins. This enzyme participates in a unique genetic code expansion process and seems essential for studying protein synthesis and engineering. Its function may be key to understanding how genetic code diversity and protein translation work in archaeal species.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
1. In Vitro Aminoacylation Assays for Pyrrolysine tRNA Charging
This recombinant pylS can be used to study how pyrrolysyl-tRNA synthetase charges tRNA under controlled laboratory conditions. Researchers might investigate the enzyme's specificity for pyrrolysine and its matching tRNA through biochemical assays that measure tRNA charging efficiency. The N-terminal 6xHis tag makes protein purification and immobilization easier for kinetic studies. These experiments would likely provide insights into the unique properties of this archaeal aminoacyl-tRNA synthetase compared to the canonical 20 synthetases.
2. Antibody Development and Immunological Studies
The purified recombinant protein can work as an antigen for generating specific antibodies against Methanosarcina barkeri pyrrolysyl-tRNA synthetase. The >85% purity level appears sufficient for immunization protocols in laboratory animals or for in vitro antibody screening applications. The 6xHis tag can be used in ELISA-based assays to monitor antibody binding specificity and affinity. Such antibodies would probably become valuable research tools for studying pyrrolysine biosynthesis pathways in archaeal systems.
3. Protein-Protein Interaction Studies Using Affinity Purification
The N-terminal 6xHis tag allows nickel-affinity based pull-down experiments to identify potential binding partners of pylS in archaeal cellular extracts. This approach might help reveal the protein networks involved in pyrrolysine incorporation and expanded genetic code machinery. The recombinant protein can be attached to nickel-charged resins and used to capture interacting proteins from Methanosarcina cell lysates. Mass spectrometry analysis of captured proteins would likely reveal novel components of the pyrrolysine translation system.
4. Comparative Biochemical Analysis of Archaeal Translation Machinery
This recombinant pylS can be used in comparative studies examining differences between archaeal and bacterial/eukaryotic aminoacyl-tRNA synthetases. Researchers might analyze substrate specificity, cofactor requirements, and enzymatic properties through various biochemical assays. The protein represents the unique 21st aminoacyl-tRNA synthetase found only in certain methanogenic archaea. Such studies could contribute to understanding the evolution and diversity of genetic code expansion mechanisms.
