@Article{,
AUTHOR = {Yan Lin, Qin Ya-Li, Bei Wei-Cheng, Yu Xiao-Lan, Huang Hong-Liang, Chen Huan-Chun},
TITLE = {Expression of an interleukin-6 - interleukin-2 fusion protein (pIL-6-IL-2) in <i>P. pastoris</i>},
JOURNAL = {European Cytokine Network},
VOLUME = {15},
YEAR = {2004},
NUMBER = {3},
PAGES = {240--246},
URL = {http://www.techscience.com/ECN/v15n3/66380},
ISSN = {1952-4005},
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<sup>th</sup> 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 × 10<sup>5</sup> U/mg and 8 × 10<sup>t5</sup> U/mg, respectively.<br/>
This report is the first description of fused porcine cytokines expressed in P. pastoris, which might be an interesting
adjuvant product for veterinary vaccines.},
DOI = {}
}