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Purification and Refolding of a Novel β-Agarase from inclusion body of E. coli

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Journal of Ocean University of China 2007年第1期6卷 80-84页

作者： ZHANG Li LU Xinzhi HAN Feng MA Cuiping YU Wengong Marine Drug and Food Institute Ocean University of ChinaQingdao 266003P.R.China

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.

关键词： β-agarase inclusion body refolding immobilized metal affinity chromatography

Refolding and Purification of Yeast Acetyl-CoA Carboxylases CT Domain Expressed as inclusion Bodies in Escherichia coli

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Chemical Research in Chinese Universities 2009年第5期25卷 690-694页

作者： YANG Xue-ying TAO Jin ZHENG Liang-yu WANG Rui-jian ZHAO Ke CAO Shu-gui Key Laboratory for Molecular Enzymology and Engineering Ministry of Education Jilin University Changchun 130021 P. R. China

Acetyl-CoA carboxylase（ACCase） is a crucial enzyme in fatty acid synthesis, by regulating the first committed step in the process. Therefore, it is a potential target for the development of new compounds against obesity or as herbicides. The cDNA encoding yeast ACCase CT domains（YCTs） from Saccharvmyces cerevisiae was amplified by RT-PCR and inserted into the vector PET28a（＋） for bacterial expression of YCT fused to N-terminal His-tag（YCT-his6）. YCTs-his6 was expressed in Escherichia coli BL21（DE3） PLys as inclusion bodies, which was solubilized in 8 mol/L urea. Ni-agarose chromatography was used to purify the inclusion bodies under denaturing condition. Correct refolding was achieved via systematic dialysis to remove the denaturant gently in the presence of 0.5 mmol/L Triton X-100. The low concentration Triton X-100 was included in the refolding buffer to increase the solubilization and enhance dimeric formation of refolding proteins. The activity of the refolded YCT-his6 was 1.2 U/rag as measured in a spectrophotometric assay using malonyl-CoA as the substrate. To our knowledge, it is the first time that the bioactive YCT-his6 has been expressed successfully in E. coli and isolated from their inclusion bodies.

关键词： Carboxyltransferase domain Expression inclusion body Refolding

On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 265-269页

作者： Xi-Qiang ZHU Su-Xia LI Hua-Jun HE Qin-Sheng YUAN State Key Laboratory of Bioreactor Engineering and Institute of Biochemistry,East China University of Science and Technology,Shanghai 200237,China State Key Laboratory of Bioreactor Engineering and Institute of Biochemistry East China University of Science and Technology Shanghai 200237 China

The EC-SOD cDNA was cloned by polymerase chain reaction （PCR） and inserted into theEscherichia coli expression plasmid pET-28a（+） and transformed into E.coli BL21 （DE3）.The correspondingprotein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactiveinclusion bodies.This structure was first solubilized under denaturant conditions （8.0 M urea）.Then,after acapture step using immobilized metal affinity chromatography （IMAC）,a gradual refolding of the protein wasperformed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione（GSH）and oxidized glutathione（GSSG）.The mass ratio of GSH to GSSG was 4:1.The purified enzyme was active,showing that at least part of the protein was properly refolded.The protein was made concentrated byultrafiltration,and then isolated using Sephacryl S-200 HR.There were two protein peaks in the A280 profile.Based on the results of electrophoresis,we concluded that the two fractions were formed by protein subunitsof the same mass,and in the fraction where the molecular weight was higher,the dimer was formed throughthe disulfide bond between subunits.Activities were detected in the two fractions,but the activity of thedimer was much higher than that of the single monomer.The special activities of the two fractions werefound to be 3475 U/mg protein and 510 U/mg protein,respectively.

关键词： refolding His6 tag recombinant EC-SOD inclusion body protein subunit structure

Expression and Purification of Soluble Human Programmed Death-1 in Escherichia coli

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Cellular & Molecular Immunology 2006年第2期3卷 139-143页

作者： Lihui Xu Yi Liu Xianhui He Institute of Bioengineering College ot Life Science and lecnnology Jinan University Guangzhou 510632 China Institute of Tissue Transplantation and Immunology College of Life Science and Technology Jinan University Guangzhou 510632 China Department of Dermatopathology Institute of Dermatology Chinese Academy of Medical Sciences and Peking Union Medical College Nanjing 210042 China

Programmed death-1 （PD-1）, a member of CD28 family, is able to negatively regulate the TCR complex-initiated signaling by interacting with its cognate ligands （PD-L1 and/or PD-L2）. PD-1/PD-L1 pathway plays an important role in down-regulating the effective phase of adaptive immune responses and the blockade of this pathway has been proved to enhance antiviral and antitumoral immunity, suggesting that it might be a potential target for the development of therapies to improve T cell responses in patients with virus infections or malignancies. In present study, the extracellular domain of human PD-1 with a carboxyl terminal His-tag （designated as sPD-1） was expressed as inclusion bodies in Escherichia coll. The product was on-column refolded, purified by immobilized metal affinity chromatography, and characterized by Western blotting. Furthermore, the soluble PD-1 with high purity possessed specific binding activity with its cognate ligand PD-L1, and the dissociation constant was 0.43 nmol/L as determined by Scatchard plot analysis. These results suggest that refolded sPD-1 from prokaryotic cells may be of therapeutic interest in enhancing antivirus and antitumoral immune responses.

关键词： PD-1 extracellular domain prokaryotic expression inclusion body binding activity

Visualized and precise design of artificial small RNAs for regulating T7 RNA polymerase and enhancing recombinant protein folding in Escherichia coli

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Synthetic and Systems Biotechnology 2016年第4期1卷 265-270页

作者： Yujia Zhao Jingjing Fan Jinlin Li Jun Li Xiaohong Zhou Chun Li Department of Biological Engineering School of Life ScienceBeijing Institute of TechnologyBeijing 10008China

Small non-coding RNAs(sRNAs)have received much attention in recent years due to their unique biological properties,which can efficiently and specifically tune target gene expressions in bacteria.Inspired by natural sRNAs,recent works have proposed the use of artificial sRNAs(asRNAs)as genetic tools to regulate desired gene that has been applied in several fields,such as metabolic engineering and bacterial physiology studies.However,the rational design of asRNAs is still a challenge.In this study,we proposed structure and length as two criteria to implement rational visualized and precise design of asRNAs.T7 expression system was one of the most useful recombinant protein expression systems.However,it was deeply limited by the formation of inclusion body.To settle this problem,we designed a series of asRNAs to inhibit the T7 RNA polymerase(Gene1)expression to balance the rate between transcription and folding of recombinant protein.Based on the heterologous expression of Aspergillus oryzae Li-3 glucuronidase in E.coli,the asRNA-antigene1-17bp can effectively decrease the inclusion body and increase the enzyme activity by 169.9%.

关键词： Artificial small RNAs Visualized and precise design Prokaryotic T7 expression system inclusion body

Oculopharyngeal Weakness, Hypophrenia, Deafness, and Impaired Vision： A Novel Autosomal Dominant Myopathy with Rimmed Vacuoles

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Chinese Medical Journal 2016年第15期129卷 1805-1810页

作者： Ting Chen Xiang-Hui Lu Hui-Fang Wang Rui Ban Hua-Xu Liu Qiang Shi Qian Wang Xi Yin Chuan-Qiang Pu Department of Neurology Chinese PLA General Hospital Beijing 100853 China Department of Neurology West China Hospital Sichuan University Chengdu Sichuan 610041 China Department of Neurology Hainan Branch of Chinese PLA General Hospital Sanya Hainan Province China

Background： Myopathies with rimnled vacuoles are a heterogeneous group of muscle disorders with progressive muscle weakness and varied clinical manifestations but similar features in muscle biopsies. Here, we describe a novel autosomal dominant myopathy with rimmed vacuoles in a large family with 11 patients of three generations affected. Methods： A clinical study including family history, obstetric, pediatric, and development history was recorded. Clinical examinations including physical examination, electromyography （EMG）, serum creatine kinase （CK）, bone X-rays, and brain magnetic resonance imaging （MRI） were performed in this family. Open muscle biopsies were performed on the proband and his mother. To find the causative gene, the whole-exome sequencing was carried out. Results： Disease onset was from adolescence to adulthood, but the affected patients of the third generation presented an earlier onset and more severe clinical manifestations than the older generations. Clinical features were characterized as dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision. However, not every patient manifested all symptoms. Serum CK was mildly elevated and EMG indicated a myopathic pattern. Brain MRI showed cerebellum and brain stem mildly atrophy. Rimmed vacuoles and inclusion bodies were observed in muscle biopsy. The whole-exome sequencing was performed, but the causative gene has not been found. Conclusions： We reported a novel autosomal dominant myopathy with rimmed vacuoles characterized by dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision, but the causative gene has not been tbund and needs further study.

关键词： inclusion body Rimmed Vacuoles Whole-exome Sequencing

Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2006年第1期7卷 13-19页

作者：龚兴国纪静谢捷周远张俊彦钟文涛 Institute of Biomacromolecule and Enzyme Engineering School of Life Sciences Zhejiang UniversityHangzhou 310027 China

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl β-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems em- ployed in earlier Src expression.

关键词： v-Src GST-fusion inclusion body Orthogonalization Protein tyrosine kinase

Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

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Chemical Research in Chinese Universities 2009年第1期25卷 64-68页

作者： XU Guang-wei WANG Jia-peng HUANG Xue-mei ZHANG Ying-jiu Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education Jilin University Changehun 130021 P R. China

To analyze whether expressed amyloid precursor protein（APP） existed in hydrophilic（cytoplasmid） or hydrophobic（lipid bilayer） environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coll. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hydrophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni^2＋ agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coli cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.

关键词： Amyloid precursor protein Expression characterization Preparation Membrane inclusion body

Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

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Animal Husbandry and Feed Science 2014年第2期6卷 55-58页

作者： Liu Xueguang Zheng Huaidong Guo Xinshuo Luo Jin Lin Cuicui Wang Qiuyu Bureau of Testing on Aquatic Products Quality Safety of Liaoning Province College of Life Sciences Liaoning University

[Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

关键词： Infectious hematopoietic necrosis virus(IHNV) Prokaryotic expression inclusion body G protein Antibody