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http://dx.doi.org/10.7845/kjm.2013.3022

Construction, Expression, and Purification of N-Terminal Variants of Lumazine Protein from Photobacterium leiognathi  

Kang, Kyoung-Suk (Department of Biochemistry, Chungnam National University)
Kim, So-Young (Department of Biochemistry, Chungnam National University)
Choi, Ji-Sun (Department of Biochemistry, Chungnam National University)
Kim, Young-Doo (Department of Biochemistry, Chungnam National University)
Pokoo, Robert (Department of Biochemistry, Chungnam National University)
Nam, Ki-Seok (Department of Biochemistry, Chungnam National University)
Lee, Chan Yong (Department of Biochemistry, Chungnam National University)
Publication Information
Korean Journal of Microbiology / v.49, no.2, 2013 , pp. 205-210 More about this Journal
Abstract
Lumazine protein is a fluorescent protein isolated from the bioluminescent bacteria of Photobacterium species. To generate minimal size of lumazine protein with possessing fluorescent characteristic, the gene coding for the wild type N-terminal domain of lumazine protein (N-LumP 118) containing amino acids up to 118 from Photobacterium leiognathi was produced. In addition, the genes coding for the variant proteins of N-LumP 118, replaced with one tryptophan amino acid (N-LumP 118 V41W, S48W, T50W, D64W, and A66W), were also constructed by Polymerase Chain Reaction and Site Directed Mutagenesis. These proteins were expressed in Escherichia coli by transformation with recombinant plasmids and purified by 6X-His tagging system. Spectroscopic studies have show that the purified proteins are capable of binding to the fluorescent ligand 6,7-dimethyl-8-ribityllumazine, resulted in showing of fluorescent characteristic with the minimal size of protein. From these studies, the mutant proteins containing single tryptophan amino acid residue, possessing its own intrinsic flouophore character at the different position, will be able to the use as a probe for further studies to deduce their three dimensional structure and the binding modes.
Keywords
Photobacterium; bioluminescence; lumazine protein; site directed mutagenesis;
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