Expression, Purification and Bioactivity of Recombinant Human Interleukin-1α Expressed in Pichia pastoris

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.035

Abstract

Abstract: This work is to secret and express human interleukin-1α(rhIL-1α)in Pichia pastoris and optimize the fermentation and purification process of rhIL-1α for obtaining the rhIL-1α with high-purity, high-expression and owing biological activity. The gene hIL-1α amplified by PCR was constructed into the eukaryotic expression vector pPICZαA/hIL-1α, and then it was transformed into the P. pastoris X-33 strain via electroporation．The engineering strain with high-expression of rhIL-1α was screened and assayed by the methods of PCR and SDS-PAGE, further indentified by Western blot. The expressed product was purified by the DEAE Sepharose Fast Flow ion exchange chromatography, and the bioactivity of it to human cancer Bel-7402 cell was initially assayed. Results showed that inducing rhIL-1α by methanol for 4 d at shaking flask level, the expression reached 30mg/L, the test by Western blot revealed that specific binding activity of rhIL-1α was detected；the purity of the rhIL-1α reached about 95％ and the yield was about 40%；rhIL-1α inhibited the proliferation of Bel-7402 cells. In conclusion, recombinant engineering vector of rhIL-1α was successfully constructed, and it was highly expressed in P. pastoris, which lays groundwork for further study of its function and bioactivity.

Key words: recombinant human interleukin-1α, Pichia pastoris, expression, purification, Bel-7402 cell

Li Bin, Xu Xiaoya, Yang Ganggang, Cui Qingqing, Yang Yajuan, Xu Cunshuan. Expression, Purification and Bioactivity of Recombinant Human Interleukin-1α Expressed in Pichia pastoris[J]. Biotechnology Bulletin, 2015, 31(9): 244-250.

References

[1] Dinarello CA. IL-1：disoveries, controversies and future directions[J]. Eur J Immunol, 2010, 40(3)：599-606.
[2] Sims JE, March CJ, Cosman D, et al．cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily[J]. Science, 1988, 241(4865)：585-589．
[3]Chizzonite R, Truitt T, Kilian PL, et al. Two high-affinity interleukin 1 receptors represent separate gene products[J]. Proc Natl Acad USA, 1989, 86(20)：8029-8033．
[4]刘海燕. 抗人IL-1α单克隆抗体及其应用：中国, 20111011425-35. 9[P]. 2011-05-30.
[5]Apte RN, Voronov E. Is interleukin-l a good or bad‘guy’in tumor immunobiology and imnmnotherapy?[J]. Immunol Rev, 2008, 222：222-241.
[6]DinareUo CA. Immunological and inflammatory functions of the interleukin-1 family[J]. Annu Rev Immunol, 2009, 27：519-550.
[7]Dinarello CA, In Durum SK, Oppenheim JJ, Feldmann M. Cytokine reference：a compendium of cytokines and other mediators of host defense[M]. Boston：Academic Press, 2001：307-318.
[8] Schoch P, Pomytkin I. A pilot study on the effect of interleukin-1 alpha on collagen deposition in aging skin[D]. Presentation abstract 12th Anti-Aging Medicine World Congress, 2014.
[9]林丹丹, 刘春亮, 包光明, 等. 小鼠IL-1α多克隆抗体的制备及应用[J]. 细胞与分子免疫学杂志, 2011, 27(9)：1007-8738.
[10]Ari H, Markus A. Intracellular functions of N-linked glycans[J]. Science, 2001, 291：2364- 2369.
[11]苟万晓, 卫红伟, 胡元森, 等. 毕赤酵母发酵生产甲醇蛋白工艺条件的优化[J]. 中国酿造, 2014, 33(7)：0254-5071.
[12]Charoenrat T, Ketudat M, Stendahl AH . Oxygen-limited fed-batch process：an altemative control for Pichia pastoris recombinant protein processes[J]. Bioproc Biosyst Eng, 2005, 27(6)：399-406.
[13]娄瑞娟, 罗立龙, 张霞, 等. 巴斯德毕赤酵母表达系统的研究进展和前景展望[J]. 生物学杂志, 2010, 27(5)：1008-9635.
[14]闵兆升, 郭会明, 颜旭, 等. 巴斯德毕赤酵母(P. pastoris)高密度发酵研究进展[J]. 生物技术通报, 2014(3)：42-49.
[15]刘红, 潘红春, 蔡绍皙, 等. 发酵条件对毕赤酵母表达重组人干扰素糖基化的影响[J]. 生物工程学报, 2005, 21(1)：107- 112.
[16]Ronanos MA, Clare JJ, Beesleyk KM, et al. Recombinant borde tella pertussis pertactin(p69)from the yeast Pichia pastoris：High-level production and immun ologica l properties[J]. Vaccine, 1991, 9：901-906.
[17]孙士青, 王少杰, 李秋顺, 等. 考马斯亮蓝法快速测定乳品中蛋白质含量[J]. 山东科学, 2011, 24(6)：6-14.
[18]Ming WP, Li MY, Dan J, et al. Inhibition of RAP1 enhances corneal recovery following alkali injury[J]. iOVS, 2015, 56(2)：711-721.
[19] Li YY, Lian HN, Jia QZ, et al. Proteome screening of pleural effusions identifies IL1A as a diagnostic biomarker for non-small cell lung cancer[J]. Biochemical and Biophysical Research Communications, 2015, 457(2)：177-182.
[20]Rider P, Carmi Y, Voronov, et al. Interleukin-1alpha[J]. Semin Immunol, 2013, 25(6)：430-438.
[21]闫学文, 单士军. 白细胞介素1α对人永生化角质形成细胞、水通道蛋白3表达的调节[J]. 中国中西医结合皮肤性病学杂志, 2012, 11(4)：211-214.
[22]李树, 王杰. 小鼠皮肤损伤修复过程中白细胞介素1α和白细胞介素1β变化与损伤时间是关系[J]. 中国组织工程研究与临床康复, 2008, 12(15)：2879-2882.
[23]Sygmund C, Gutmann A, Krondorfer I, et al. Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris[J]. Appl Microbiol Biotechnol, 2012, 94(3)：695-704.
[24]王治, 郑甲, 唐诗哲, 等. 外源蛋白在巴斯德毕赤酵母中高效分泌表达的前沿技术[J]. 基因组学与应用生物学, 2014, 33(3)：689-694.
[25]Zhu T, You L, Gong F, et al. Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production of alkaline β-mannanase in Pichia pastoris[J]. Enzyme and Microbial Technology, 2011, 49(4)：407-412.
[26]高敏杰, 史仲平. 甲醇营养型重组毕赤酵母高效表达外源蛋白过程的控制与优化[J]. 中国化学工程学报, 2013, 21(2)：216-226.
[27]?elik E, ?al?k P, Oliver SG. Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources：Effects of methanol feeding rate[J]. Biotechnology and Bioengineering, 2010, 105(2)：317-329.
[28]Swati A, Deepali C, Vijay C, et al. Polymorphisms in IL-1 gene cluster and its association with the risk of perinatal HIV transmission, in an Indian cohort[J]. Immunology Letters, 2013, 153：1-8.
[29]Georgia P, Hilary ODC, Stephen GH, et al. IL1 A and IL4 signalling in human ovarian surface epithelial cells[J]. Journal of Endocrinology, 2011, 211：273-283.