Optimization of the secretory expression of recombinant human C-reactive protein in Pichia pastoris
Optimization of the secretory expression of recombinant human C-reactive protein in Pichia pastoris
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重组人C反应蛋白在毕赤酵母中分泌表达的优化
DOI：
10.1007/s13205-017-0917-0
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发表时间：
2017-08
期刊：
影响因子：
2.8
通讯作者：
Hu Xuejun
中科院分区：
文献类型：
--
作者：
Li Junming;Sun Chengming;Chen Lei;Sun Lihui;Duan Lijun;Zheng Qing;Hu Xuejun
人C反应蛋白（CRP）是一种经典的急性时相血浆蛋白，不仅是一种敏感的全身炎症标志物，而且是心血管疾病的独立危险预测因子。然而，现有的用于表达CRP的异源表达系统对于CRP的大规模工业化生产以满足对CRP的不断增长的市场需求而言是不高效且不划算的。本研究旨在通过对信号肽、启动子和碳源的优化来提高重组CRP在毕赤酵母中的分泌。设计并合成了编码四种不同信号肽的CRP基因。采用重叠延伸聚合酶链反应（SOE-PCR）技术，将其分别克隆到pPICZαA和pPICZ B中，并在醇氧化酶I启动子（pAOX 1）的调控下，在毕赤酵母X-33中表达。在这些信号肽中，由α-交配因子分泌信号肽（α-MF）主导的CRP分泌量最高。然后，通过切换到甘油醛-3-磷酸脱氢酶启动子（pGAP）实现CRP基因的组成型构建和表达。随后，使用不同的碳源和不同的浓度，以进一步提高CRP的分泌。用pGAP驱动的α-MF表达CRP得到了最高的分泌型CRP产量，在优化的培养条件下约为3 mg/l培养物。用ELISA法检测纯化的重组CRP与抗人CRP单克隆抗体具有良好的免疫反应性。
Human C-reactive protein (CRP), a classical human acute-phase plasma protein, is not only a sensitive systemic inflammatory marker but also an independent risk predictor of cardiovascular diseases. However, existing heterologous expression systems for expressing CRP is not efficient and cost-effective for large-scale industrial production of CRP to meet the growing market demand for CRP. This study aims to improve the secretion of recombinant CRP by Pichia pastoris via optimizing signal peptides, promoters and carbon sources. The CRP genes with encoding four different signal peptides were designed and synthesized. The genes were cloned into pPICZαA or pPICZ B, respectively via splicing by overlap extension polymerase chain reaction (SOE-PCR) technology and expressed in P. pastoris X-33, regulated by the alcohol oxidase I promoter (pAOX1). The CRP led by the α-mating factor secretion signal peptide (α-MF) was secreted at the highest level in these signal peptides. Then, a constitutive construct and expression of the CRP genes were achieved by switching to the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). Subsequently, different carbon sources and at different concentrations were used to further improve the secretion of CRP. The expression of CRP with the α-MF driven by the pGAP gave the highest yield of secreted CRP, about 3 mg/l of culture on the optimized culture conditions. The purified recombinant CRP exhibited good immunoreactivity determined by ELISA with anti-human CRP monoclonal antibody.
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影响因子：
1.6
作者：
H. Dortay;Sandra M. Schmöckel;J. Fettke;B. Mueller‐Roeber
通讯作者：
H. Dortay;Sandra M. Schmöckel;J. Fettke;B. Mueller‐Roeber
影响因子：
6.4
作者：
Várnai A;Tang C;Bengtsson O;Atterton A;Mathiesen G;Eijsink VG
通讯作者：
Eijsink VG
影响因子：
5
作者：
McWhorter, VC;Ford, LC;Butch, AW
通讯作者：
Butch, AW
DOI：
10.1007/978-1-61779-228-1_27
发表时间：
2011-01-01
期刊：
MOLECULAR METHODS FOR EVOLUTIONARY GENETICS
影响因子：
--
作者：
Nelson, Matthew D.;Fitch, David H. A.
通讯作者：
Fitch, David H. A.
影响因子：
2.8
作者：
C. Kindmark;Jim C. Williams
通讯作者：
C. Kindmark;Jim C. Williams