The polyphenol oxidase (PPO) cds sequence was amplified by PCR from the genome of Yingshuang tea plant with the high fidelity polymerase pfu. The cds sequence was recombinanted into the eukaryotic expression vector pPICZa with the designed primer, and the fusion protein eukaryotic expression vector of tea polyphenol oxidase “pPICZa-PPO” was succeeded to be constructed, and then it was transformed into Pichia pastoris GS115. Therefore, several positive converters were screened. After those positive converters were induced by methanol,the target protein was successfully detected in the culture medium by using Western-Blotting method. The detection result of enzyme activity showed that the induced products had a relatively high enzyme activity and can correctly play their biological activity.
WANG Nai-dong
,
ZHANG Li-xia
,
HUANG Xiao-qin
,
HAN Xiao-yang
,
LI Zhi
. Construction and Expression of Fusion Protein Eukaryotic Expression Vector of Polyphenol Oxidase Gene of Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2012
, 32(3)
: 269
-275
.
DOI: 10.13305/j.cnki.jts.2012.03.012
[1] 王乃栋, 张丽霞, 向勤锃, 等. 茶树多酚氧化酶基因的生物信息学分析及原核表达[J]. 茶叶科学, 2011, 31(1): 341-347.
[2] Armstrong N, de Lencastre A, Gouaux E. A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase[J]. Protein Sci, 1999, 8(7): 1475-1483.
[3] Liu J W, Huang Y Y, Ding J, et al. Prokaryotic expression and purification of Camellia sinensis polyphenol oxidase[J]. J Sci Food Agric, 2010, 90(14): 2490-2494.
[4] Yi-Liangwu, Pan L, Yu S, et al. Cloning, microbial expression and structure activity relationship of polyphenol oxidases from Camellia sinensis[J]. Journal of Biotechnology, 2010, 145(1): 66-72.
[5] Summers C A, Flowers R A N. Protein renaturation by the liquid organic salt ethylammonium nitrate[J]. Protein Sci, 2000, 9(10): 2001-2008.
[6] 杨梅, 温真, 林丽玉, 等. 毕赤酵母蛋白表达系统研究进展[J]. 生物技术通报, 2011, 20(4): 45-47.
[7] Vedvick T S.Gene expression in yeast: Pichia pastoris[J]. Curr Opin Biotechnol, 1991, 2(5): 742-745.
[8] 戴秀玉. 一种值得注意的基因表达系统—巴斯德毕赤氏酵母[J]. 微生物学报, 1997, 37(6): 483-485.
[9] Gellissen G, Hollenberg C P.Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis: a review[J]. Gene, 1997, 190(1): 87-97.
[10] Chang P Y, Fong M Y, Nissapatorn V, et al. Evaluation of Pichia pastoris-expressed recombinant rhoptry protein 2 of Toxoplasma gondii for its application in diagnosis of toxoplasmosis[J]. Am J Trop Med Hyg, 2011, 85(3): 485-489.
[11] Siman P, Blatt O, Moyal T, et al. Chemical synthesis and expression of the HIV-1 Rev protein[J]. Chem biochem, 2011, 12(7): 1097-1104.
[12] 董双涛, 李宝霞, 霍乃蕊. TCA-丙酮沉淀法提取聚合草叶蛋白的研究[J]. 世界中西医结合杂志, 2011, 6(9): 766-767.
[13] 张毅, 屈贤铭, 杨胜利. 融合蛋白基因工程应用及研究[J]. 生物工程进展, 2000, 20(3): 13-18.
[14] 王阿慧, 孙军, 毕昊, 等. 6His-VP3融合蛋白的表达、纯化及其捕获细胞中相关蛋白的初步探讨[J]. 医学分子生物学杂志, 2007(2): 112-115.
[15] Kato T, Park E Y.Specific expression of GFPuvβ-1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide[J]. Biochemical and Biophysical Research Communications, 2007, 359(3): 543-548.
[16] 刘劫, 马大龙. 新型双分子细胞因子融合蛋白研究进展[J]. 生物化学与生物物理进展, 1994, 21(3): 194-196.
[17] Pandjaitan B, Eibensteiner P B, Vrtala S, et al. pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens[J]. Gene, 1999, 237(2): 333-342.
[18] Strugnell S A, Wiefling B A, Deluca H F.A modified pGEX vector with a C-terminal histidine tag: recombinant double-tagged protein obtained in greater yield and purity[J]. Anal Biochem, 1997, 254(1): 147-149.
[19] Agarwal S, Jha S, Sanyal I, et al. Expression and purification of recombinant human alpha1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity[J]. J Biotechnol, 2010, 147(1): 64-72.
[20] Blanchard V, Gadkari R A, George A V, et al. High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains--selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG[J]. Glycoconj J, 2008, 25(3): 245-257.
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