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Expression,purification and Identification of Recombinant Mouse Interleukin 21 protein in ***

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Cellular & Molecular Immunology 2006年第4期3卷 311-315页

作者： Weiguo Tang Guobing Chen Qing Gu Jing Pan Wutong Wu School of Life Science and Technology China Pharmaceutical University Nanjing 210009 China Jinling Pharmaceutical Co. Ltd. NanJing 210009 China

Interleukin 21 （IL-21） is a novel type I cytokine that is significantly homologous to IL-2, IL-4 and IL-15. Its receptor complex contains γc chain which is also a component of receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, so there may be overlapping or relevancies in their biological functions. IL-21 is capable of co-stimulating mature T cells, B cells, NK cells, and of stimulating CD16 expression on the surface of NK cells to induce ADCC in innate immune response. It can also strengthen the anti-tumor effect of the cellular immunity, especially v/a enhancing the activities of NK and antigen specific CTL cells. Thus, IL-21 is a potential useful therapeutic molecule for immunotherapy of malignancies, by eliciting innate and adaptive anti-tumor immune responses in tumor-bearing hosts. In order to study the biological functions of IL-21, we constructed a mIL-21 prokaryotic expression plasmid and expressed the recombinant mIL-21 protein in E. coli in present study. The recombinant plasmid pET28a/mIL-21 with a carboxyl terminal His-tag was subcloned from the pcDNA3.1/mIL-21 and expressed in E. coli. The induced protein was detected by SDS-PAGE, and identified by Western-blot assay with anti-mIL-21 antibody. The recombinant protein was purified v/a Ni^＋ affinity chromatography, and renatured with GSH/GSSG system. Our mouse T cell proliferation experiment showed that the recombinant mIL-21 protein could enhance the mouse T cell proliferation either by itself alone or in the presence of Con A.

关键词： interleukin 21 gene expression protein purification

Extraction, purification and Identification of Bone Morphogenetic protein in Conditioned Medium of Osteosarcoma Cell (MG-63)

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The Chinese-German Journal of Clinical Oncology 2003年第4期2卷 234-236,253页

作者：吴华冷燕奎陈安民翟智玲陈继革武汉华中科技大学同济医学院附属同济医院 430030

Objective: To find out a method of extraction and purification of bone morphogenetic protein (BMP) from osteosarcoma cell conditioned medium, and evaluate the biological activity of ***: Conditioned medium of osteosarcoma cell lines (MG-63) was collected, concentrated and dialyzed. The concentrated protein was purified through gel chromatography on Sephcryl-S-100. The purified protein was tested by BMP monoclonal antibody (McAb), its molecular weight (MW) was determined by SDS-PAGE and its biological activity was demonstrated by heterotopic ***: The purified protein was proved to be BMP by BMP McAb, had a satisfactory heterotopic ossification, and its MW was about 21 ***: BMP existed in the conditioned medium of osteosarcoma cell and had a satisfactory biological activity after purification. Because osteosarcoma cell can be cultured and grew for a long timein vitro, this method will be helpful to a vast extraction of BMP and clinical application. Key words osteosarcoma cell - conditioned medium - bone morphogenetic protein - protein purification This project was a key scientific and technological program of Hubei Provicial Scientific and Technological Committee (No. 002p1503).

关键词： osteosarcoma cell conditioned medium bone morphogenetic protein protein purification

Expression and purification of Serine/Arginine-Rich Splicing Factor 1 from Escherichia coli Expression System

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Journal of Donghua University(English Edition) 2022年第5期39卷 441-445页

作者： ZHANG Minmin ZHANG Yunlong CHEN Ting LU Changrui College of Biological Science and Medical Engineering Donghua UniversityShanghai 201620China

Serine/arginine-rich splicing factor 1(SRSF1), as a prototype member of the highly conserved serine/arginine family of RNA binding proteins, plays an important role in mRNA alternative splicing, stabilization, nuclear export, and translation. Here, the expression system was established to purify full-length human SRSF1 from Escherichia coli(E. coli). The SRSF1 coding sequence was amplified by polymerase chain reaction(PCR) and inserted into the pET-28 a-ppSUMO vector with His-tag to construct a recombinant plasmid His-SUMO-SRSF1. Then the plasmid was transformed into BL21(DE3) competent cells for expression. After purification by affinity chromatography and cleavage of His-SUMO moiety, a highly purified SRSF1 with a molecular weight of around 28 kg/mol was obtained. The protein was analyzed by sizing chromatography and it was found that SRSF1 would form a polymer structure in the solution. According to Expasy bioinformatics analysis, SRSF1 is extremely unstable. purification of full-length SRSF1 protein provides an opportunity to study mRNA splicing in vitro.

关键词： protein purification serine/arginine-rich splicing factor 1(SRSF1) SUMO SUMO protease bioinformatics chromatography

Optimization of DsbA purification from Recombinant Escherichia coli Broth Using Box-Behnken Design Methodolog

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Chinese Journal of Chemical Engineering 2013年第2期21卷 185-191页

作者： LUO Man GUAN Yixin YAO Shanjing Department of Chemical and Biological Engineering Zhejiang University Hangzhou 310027 China

Disulfide bond formation protein A （DsbA） is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was （377.9±1.7） mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gil2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.

关键词： disulfide bond formation protein A protein purification Box-Behnken experiment design response surface methodology multi-object programming

purification and Characterization of Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase from the Dromedary Camel

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Acta Biochimica et Biophysica Sinica 2007年第2期39卷 148-154页

作者： Latifa FOURRAT,Abdelghani IDDAR,Abdelaziz SOUKRI 1Laboratoire de Physiologie et Génétique Moléculaire Département de Biologie Facultédes Sciences A■n-Chock UniversitéHassan-Ⅱ. Casablanca Morocco ~2Centre National de l'Energie des Sciences et des Techniques Nucléaires Center d'Etudes Nucléaires de la Ma■mora Rabat 10001 Morocco

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH）（EC 1.***.1.12）,a key enzyme ofcarbon metabolism,was purified and characterized to homogeneity from skeletal muscle of *** protein was purified approximately 26.8 folds by conventional ammonium sulphatefractionation followed by Blue Sepharose CL-6B chromatography,and its physical and kinetic propertieswere *** native protein is a homotetramer with an apparent molecular weight of approximately146 *** focusing analysis showed the presence of only one GAPDH isoform with an isoelectricpoint of *** optimum pH of the purified enzyme was *** on the effect of temperature onenzyme activity revealed an optimal value of approximately 28-32 ℃ with activation energy of 4.9 kcal/*** apparent Kmvalues for NAD+and DL-glyceraldehyde-3-phophate were estimated to be 0.025±0.040mM and 0.21±0.08 mM, respectively. The Vmaxof the purified protein was estimated to be 52.7±5.9 U/*** kinetic parameter values were different from those described previously, reflecting protein differencesbetween species.

关键词： Camelus dromedarius glyceraldehyde-3-phosphate dehydrogenase protein purification skeletal muscle

Highly Efficient and Economical Baculovirus Expression System for Preparing Human Papillomavirus Type16 Virus-like Particle

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 548-552页

作者： Jin ZHENG, Jun MA, Xiao-Feng YANG, Hong-Li LIU, Hong-Wei CHENG, Lu-Sheng SI, and Yi-Li WANGThe Key Laboratory of Biomedical Information Engineering of Ministry of Education, Institute of Cancer Research,School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710061, China The Key Laboratory of Biomedical Information Engineering of Ministry of Education Institute of Cancer Research School of Life Science and Technology Xi’an Jiaotong University

To improve the existing human papillomavirus type16 (HPV16) virus-like particle (VLP) preparation, a highly efficient, economical and timesaving system was established. Sf-9 cells were infected with recombinant baculovirus containing the target gene encoding HPV16L1 protein with 6×His tag, and harvested 72 h postinfection (p.i.) at 27 °C. The ProBondTM purification system was used for protein purification. The molecular weight of expressed HPV16L1 protein was 58 kD as revealed by SDS-PAGE, and confirmed by Western blot. The purity of denatured and native HPVL1 proteins that were prepared were 91.9% and 71.5%, respectively, which corresponded to a yield of 2.26 mg denatured protein and 1.84 mg native protein per 2×107 cells. The proteins were further analyzed by mouse erythrocyte hemagglutination assay and hemagglutination inhibition assay, and there effects on VLP formation were also visualized by transmission electron microscopy. Results showed that the native protein purified was biologically active as natural HPVL1 protein, inducing the murine erythrocyte agglutination and VLP formation. In addition, the purified recombinant HPV16L1 native protein with 6×His tag could self-assemble into virions in vitro. Hopefully, the present expression and purification system is promising to be convenient, timesaving and economical for preparation of HPV16 VLP vaccine.

关键词： human papillomavirus HPV16L1 protein purification virus-like particle (VLP)

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

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Chinese Journal of Chemical Engineering 2001年第3期9卷 314-318页

作者：张天喜刘会洲陈家镛 Chinese Acad Sci Inst Chem Met Lab Separat Sci & Engn Beijing 100080 Peoples R China

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

关键词： reversed micelles yeast alcohol dehydrogenase protein purification affinity technology cetyltrimethy- lammonium bromide

Verticase:a Fibrinolytic Enzyme Produced by Verticillium sp,Tj33,an Endophyte of Trachelospermum jasminoides

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Journal of Integrative Plant Biology 2007年第11期49卷 1548-1554页

作者： Ying Li Jing-Lei Shuang Wei-Wei Yuan Wu-Yang Huang Ren-Xiang Tan College of Horticulture Nanjing Agricultural University Nanjing 210095 China Institute of Functional Biomolecules Nanjing University Nanjing 210093 China

Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of VerticUlium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pl of 8.5, was demonstrated to be homogeneous by sodium dodecyl suIfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9-10 and 50-60℃, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease.

关键词： fibrinolytic serine protease plant endophyte protein purification Trachelosperrnum jasminoides verticase.

High-level expression, purification and characterization of codon-optimized recombinant hemagglutinin 5 proteins in mammalian cells

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Chinese Medical Journal 2010年第8期123卷 1073-1077页

作者： YANG Jing-lin WANG Hong-liang WANG Shun-xin YANG Peng LIU Kang-tai JIANG Cheng-yu National Key Laboratory of Medical Molecular Biology Institute of Basic Medical Sciences Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100005 China

Background Numerous Asian cases of avian influenza virus infection, especially the highly pathogenic strain H5N1, in humans have raised the concern that another influenza pandemic is close. However, there are no effective therapeutic drugs or preventative vaccines available. Hemagglutinin is the membrane glycoprotein of avian influenza virus responsible for receptor binding to human cells and the main immunogenic protein that elicits a strong immune response. Although this protein is of great importance to the study of pathogenesis and vaccine development, its expression and purification are difficult due to high levels of glycosylation. Methods In this study, we expressed codon-optimized, full-length hemagglutinin 5 （H5） protein fused with a human IgG Fc tag （H5-Fc） in HEK293 cells. To enhance secretion of this protein, we also deleted the transmembrane domain and the intracellular domain of the H5 protein （H5ATM-Fc）. Purified proteins were obtained using a protein A column. Results ELISA revealed that the yield of soluble H5ATM-Fc protein in the supernatant was about 20 mg/L. Western blotting and fluorescence activated cell sorter （FACS） indicated that the purified H5 protein was correctly folded and biologically active. Conclusion purification of H5 proteins from mammalian cells could be used for large-scale production of recombinant H5 protein for basic scientific research or the development of vaccines.

关键词： hemagglutinin avian influenza protein purification mammalian cell expression