Year-end Sale % OFF 
Abstract
The transformation of the strain DH5α TM-T1R with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E. coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA. 28 mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with KDs different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit.
Highlights
Selenium may be incorporated into a protein body as an amino acid selenocysteine
In the present report we describe the overexpression and purification strategy for preparation of large quantities of soluble recombinant selenophosphate synthetase (SPS) from E. coli and its binding stoichiometry to TNP-ATP in order to determine its functional activity
Selenophosphate serves as a universal selenium donor in cellular processes both in prokaryotes and eukaryotes, and its formation is catalyzed by selenophosphate synthetase (SPS), a selD gene product [23]
Summary
Selenium may be incorporated into a protein body as an amino acid selenocysteine. Selenophosphate synthetase (EC 2.7.9.3.), a product of selD gene in bacteria, catalyzes selenophosphate formation with the help of ATP and Mg2+ ions [1]. This reaction appears common for all living organisms and serves to transform selenium into biologically active form—selenophosphate. Journal of Amino Acids used as a model for studies of SPS function and properties regarding to its high structural similarity—55% [7]. The functionally active fraction of SPS was monitored following the complex formation with a fluorescent nucleotide analog for ATP, TNP-ATP. In the present report we describe the overexpression and purification strategy for preparation of large quantities of soluble recombinant SPS from E. coli and its binding stoichiometry to TNP-ATP in order to determine its functional activity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
