| Citation: |
BI Xiaomin, HUANG Guiju, FAN Sigang, ZHU Wenjie, YU Dahui. Optimization of expression conditions for recombinant protein of biomineral gene Pearlin from pearl oyster (Pinctada fucata)[J]. South China Fisheries Science, 2015, 11(6): 100-106. DOI: 10.3969/j.issn.2095-0780.2015.06.014
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To obtain high and stable expression recombinant fusion protein of Pearlin, we constructed the recombinant vector by using the open reading frames (ORF) of Pearlin cloned from the mantle tissue of pearl oyster (Pinctada fucata) and optimized the expression conditions. The Pearlin ORF was cloned into the vector pET32a and the plasmids of pET32a-Pearlin were obtained and then transformed into E.coli BL21(DE3). His-tagged insoluble fusion protein was highly expressed and the molecular weight of the fusion protein was about 34.19 kD. We optimized the conditions for IPTG concentrations, induction duration and timing, and temperature and pH of the medium. The results showed that the optimal IPTG concentration ranged from 0.6 mmol·L-1 to 1.4 mmol·L-1 and the best temperature was 37 ℃ when IPTG concentration was 1.0 mmol·L-1 and incubation time was 6 h. The expression levels of recombinant protein did not change significantly when the pH of medium was 6.0, 7.0 and 8.0. When IPTG concentration was kept constant (1.0 mmol·L-1), 4~6 h induction was optimal; when IPTG concentration (1.0 mmol·L-1) and induction duration (6 h) were kept constant, the best starting time for induction was 3~4 h after transformation. Solubility test indicates that fusion protein pET32a-Pearlin was mainly in the form of inclusion body.
| [1] |
薛桂英, 郭奕惠, 黄桂菊, 等. 合浦珠母贝不同壳基质蛋白基因的表达水平比较[J]. 广东农业科学, 2013(17): 140-141. doi: 10.3969/j.issn.1004-874X.2013.17.043
|
| [2] |
刘晓军, 李家乐. 养殖珍珠贝贝壳基质蛋白研究进展[J]. 上海海洋大学学报, 2013, 22(2): 200-204. https://www.zhangqiaokeyan.com/academic-journal-cn_detail_thesis/02012103217381.html
|
| [3] |
王小玉, 喻达辉, 黄桂菊, 等. 合浦珠母贝3个家系的AFLP标记分离与遗传多样性研究[J]. 南方水产, 2007, 3(5): 54-60. doi: 10.3969/j.issn.2095-0780.2007.05.009
|
| [4] |
郭奕惠, 黄桂菊, 喻达辉. 合浦珠母贝DNA的抽提和RAPD反应体系的优化[J]. 南方水产, 2006, 2(4): 59-64. doi: 10.3969/j.issn.2095-0780.2006.04.010
|
| [5] |
喻达辉, 王小玉, 郭奕惠, 等. RAPD标记在合浦珠母贝家系F1代的分离方式[J]. 南方水产, 2005, 1(6): 1-7. doi: 10.3969/j.issn.2095-0780.2005.06.001
|
| [6] |
ISOWA Y, SARASHINA I, DAVIN H E, et al. A comparative study of the shell matrix protein Aspein in Pterioid Bivalves[J]. J Mol Evol, 2012, 75(1/2): 11-18. doi: 10.1007/s00239-012-9514-3
|
| [7] |
LIU X J, LIU C, CHEN L, et al. A new method to extract matrix proteins directly from the secretion of the mollusk mantle and the role of these proteins in shell biomineralization[J]. Mar Biotechnol, 2011, 13(5): 981-991. doi: 10.1007/s10126-011-9362-y
|
| [8] |
LIU X J, LIU C, SUN J, et al. Erosion of the prismatic layer by the organic matrix during the formation of the nacre-prism transition layer in the shell of Pinctada fucata (Bivalvia, Mollusca) [J]. Mar Biol, 2011, 56 (9): 869-876. doi: 10.1007/s11434-011-4348-8
|
| [9] |
SONG X R, WANG X T, LI L, et al. Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas[J]. Mol Biol Rep, 2014, 41(7): 4273-4278. doi: 10.1007/s11033-014-3298-z
|
| [10] |
JOUBERT C, PIQUEMAL D, MARIE B J, et al. Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization[J]. BMC Genomics, 2010, 11: 613. doi: 10.1186/1471-2164-11-613
|
| [11] |
MARIE B J, ZANELLA I, GUICHARD N, et al. Novel proteins from the calcifying shell matrix of the Pacific oyster Crassostrea gigas[J]. Mar Biotechnol, 2011, 13(6): 1159-1168. doi: 10.1007/s10126-011-9379-2
|
| [12] |
闫振广. 合浦珠母贝贝壳珍珠层形成机理的研究[D]. 北京: 清华大学, 2008: 1-12. https://cdmd.cnki.com.cn/Article/CDMD-10003-2009083414.htm
|
| [13] |
YU J, WANG H, DU X D, et al. Dermatopontin, a shell matrix protein gene from pearl oyster Pinctada martensii, participates in nacre formation [J]. BBRC, 2012, 425(3): 679-683. doi: 10.1016/j.bbrc.2012.07.099
|
| [14] |
ZHANG Y, XIE L P, MENG Q X, et al. A novel matrix protein participating in the nacre framework formation of pearl oyster Pinctada fucata [J]. CBP, 2003, 135(3): 565-573. doi: 10.1016/S1096-4959(03)00138-6
|
| [15] |
ZHANG L J, HE M X. Quantitative expression of shell matrix protein genes and their correlations with shell traits in the pearl oyster Pinctada fucata[J]. Aquaculture, 2011, 314(1/2/3/4): 73-79. doi: 10.1016/j.aquaculture.2011.01.039
|
| [16] |
WANG N, KINOSHITA S, RIHO C, et al. Quantitative expression analysis of nacreous shell matrix protein genes in the process of pearl biogenesis[J]. CBP, 2009, 154(3): 346-350. doi: 10.1016/j.cbpb.2009.07.012
|
| [17] |
ZHANG C, XIE L P, HUANG J, et al. A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata[J]. BBRC, 2006, 344(3): 735-740. doi: 10.1016/j.bbrc.2006.03.179
|
| [18] |
MIYASHITA T, TAKAGI R, OKUSHIMA M, et al. Complementary DNA cloning and characterization of Pearlin, a new class of matrix protein in the nacreous layer of oyster pearls[J]. Mar Biotechnol, 2000, 2(5): 409-418. doi: 10.1007/PL00021687
|
| [19] |
MATSUSHIRO A, MIYASHITA T, MIYAMOTO H, et al. Presence of protein complex is prerequisite for aragonite crystallization in the nacreous layer[J]. Mar Biotechnol, 2000, 5(1): 37-44. doi: 10.1007/s10126-002-0048-3
|
| [20] |
CHECA A. A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca)[J]. Tissue and Cell, 2000, 32(5): 405-416. doi: 10.1054/tice.2000.0129
|
| [21] |
GREGOIRE C. Topography of the organic components in mother of pearl [J]. JBBC, 1957, 3(5): 797-808. doi: 10.1083/jcb.3.5.797
|
| [22] |
BLAY C, KOUA M S, VONAU V. Influence of nacre deposition rate on cultured pearl grade and colour in the black-lipped pearl oyster Pinctada margaritifera using farmed donor families[J]. Aquac Int, 2014, 22(2): 937-953. doi: 10.1007/s10499-013-9719-5
|
| [23] |
INOUE N, ISHIBASHI R, SHIKAWA T, et al. Comparison of expression patterns of shell matrix protein genes in the mantle tissues between high-and low-quality pearl-producing recipients of the pearl oyster, Pinctada fucata[J]. Zool Sci, 2011, 28(1): 32-36. doi: 10.2108/zsj.28.32
|
| [24] |
INOUE N, ISHIBASHI R, ISHIKAWA T, et al. Can the quality of pearls from the Japanese pearl oyster (Pinctada fucata) be explained by the gene expression patterns of the major shell matrix proteins in the pearl sac [J]. Mar Biotechnol, 2011, 13(1): 48-55. doi: 10.1007/s10126-010-9267-1
|
| [25] |
龙敏明, 黄桂菊, 邹记兴, 等. 育珠对合浦珠母贝N19和Prismalin-14基因表达水平的影响[J]. 南方水产科学, 2013, 9(5): 58-63. doi: 10.3969/j.issn.2095-0780.2013.05.010
|
| [26] |
BERLEC A, TOMPA G, SLAPAR N, et al. Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis[J]. LAM, 2008, 46(2): 227-231. doi: 10.1111/j.1472-765X.2007.02297.x
|
| [27] |
金晶, 蔡亦红, 类延花, 等. HCMVpp65截短蛋白原核表达条件优化[J]. 微生物学杂志, 2005, 25(3): 28-32. doi: 10.3969/j.issn.1005-7021.2005.03.008
|
| [28] |
邹平. 重组包涵体蛋白质复性[J]. 厦门科技, 2005(5): 42-45.
|
| [29] |
王晓霞. 绵羊痘病毒甘肃株两种糖蛋白基因的克隆表达及间接ELISA诊断方法的建立[D]. 杨凌: 西北农林科技大学, 2009: 35-36.
|
| [30] |
祝珍珍. 山羊△FosB基因的原核表达、多克隆抗体制备及组织差异表达检测[D]. 杨凌: 西北农林科技大学, 2010: 27.
|
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