Open Access

ARTICLE

Expression of an interleukin-6 - interleukin-2 fusion protein (pIL-6-IL-2) in P. pastoris

Yan Lin, Qin Ya-Li, Bei Wei-Cheng, Yu Xiao-Lan, Huang Hong-Liang, Chen Huan-Chun^*

Laboratory of Animal Virology, College of Animal Science, Veterinary Medicine, Huazhong Agricultural University, 1 Shi Zi Shan st, Hong Shan District, Wuhan, Hubei 430070, China

* Corresponding Authors: Chen Huan-chun, ;

European Cytokine Network 2004, 15(3), 240-246.

Abstract

Interleukin-2 and interleukin-6 can stimulate the growth and proliferation of T lymphocytes and the differentiation of activated B lymphocytes respectively, and in turn enhance cellular and humoral immune responses. In this work, an expression clone using Pichia pastoris, a methylotrophic yeast strain, has been developed in order to produce large amounts of the functional recombinant fusion protein pIL-6-IL-2, which contains the mature porcine interleukin-6 peptide and the mature porcine interleukin-2 peptide. Two components of the fusion protein were connected by means of a flexible linker (Gly-Gly-Gly-Gly-Ser-Glu-Phe-Gly-Ser-Gly-Gly). In response to 1% methanol induction, the recombinant strain GS115/9K-IL6-IL2 secreted an exogenous protein, with a molecular weight of approximately 40 kD, into the culture medium. This was confirmed to be pIL-6-IL-2 by means of SDS-PAGE and Western Blot analysis. The protein was visible on the 2nd day following methanol induction, and peaked on the 4^th day. By this time, the level had reached 50 mg/L as determined using the method of Bradford. After treatment with PNGase F and analysis of the concentration of sugar, the fusion protein pIL-6-IL-2 was further confirmed to be mainly a glycoprotein with an approximately 2 kDa sugar decoration. In addition, the IL-6 and IL-2 biological activities of the fusion protein, determined by cell proliferation assays using the IL6-dependent cell line B9 and the IL2-dependent cell line CTLL-2, reached 1 × 10⁵ U/mg and 8 × 10^t5 U/mg, respectively.
This report is the first description of fused porcine cytokines expressed in P. pastoris, which might be an interesting adjuvant product for veterinary vaccines.

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