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Obelin hyperexpression in E. coli, purification and characterization
SS Markova2; ES Vysotski2; John Lee*1
(1) Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA; (2) Institute of Biophysics, RAS (SB), 660036 Krasnoyarsk, Russia, and University of Georgia, USA *(firstname.lastname@example.org)
Hyperexpression of obelin from Obelia longissima was obtained using the pET expression system. The nucleotide sequence coding apoobelin was amplified from cDNA by PCR and directly subcloned into the ATG start codon of pET19b. The new restriction sites for direct cloning were generated by design of the primers. Replacement of Ser for Ala in the second position of the obelin sequence was a consequence of the incorporation of the NcoI site at the translation initiation codon. The final plasmid was transformed in E. coli strain BL21-Gold (DE3). The recombinant apoobelin from the inclusion bodies was obtained highly purified after two chromatography runs. The protein in 6 M urea was refolded by 10-fold dilution into a Tris-EDTA buffer with a slight excess of coelenterazine. The photoprotein product was homogeneous according to LC-Electrospray IMS. As shown by N-terminal a/a sequence, the Met-1 is digested on producing recombinant apoobelin in E. coli cells. The yield of high purity obelin is 75 mg per liter of LB and 30 mg per liter in defined media (for NMR study). Highest quality protein is desirable for obtaining good crystals. Some spectral characteristics of recombinant obelin are described. Supported by ONR and RAS.
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