Expression of an interleukin-6 - interleukin-2 fusion protein (pIL-6-IL-2) in P. pastoris.
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 4th 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 x 10(5) U\mg and 8 x 10(5) 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.
Lin
Yan
Y
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.
Ya-Li
Qin
Q
Wei-Cheng
Bei
B
Xiao-Lan
Yu
Y
Hong-Liang
Huang
H
Huan-Chun
Chen
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D015850:Interleukin-6 / Q000235:genetics*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013552:Swine
D017931:DNA Primers; D007376:Interleukin-2; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase