Preparation of Functionally Active Recombinant Human Interleukin-6

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V. A. Spiridonova^1*, A. S. Lygina², M. M. Anohina³, and N. N. Tupitsyn³

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: spiridon@belozersky.msu.ru

²Chemical Faculty, Lomonosov Moscow State University, 119992 Moscow, Russia; E-mail: lygina@mail.ru

³Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe Shosse 24, 115478 Moscow, Russia; fax: (495) 324-1430; E-mail: cannt@aha.ru

^* To whom correspondence should be addressed.

Received October 10, 2006; Revision received December 8, 2006

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The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.

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KEY WORDS: interleukin-6, cloning, highly active protein, ELISA, gp130 receptor dimerization

DOI: 10.1134/S0006297907040098

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