1.Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein.
Xiaoli SONG ; Xiaoyun LIU ; Lei CAI ; Jianwei WU ; Jihua WANG
Journal of Biomedical Engineering 2015;32(6):1267-1272
In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.
Escherichia coli
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Freeze Drying
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Humans
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Recombinant Fusion Proteins
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biosynthesis
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Troponin C
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biosynthesis
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Troponin I
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biosynthesis
2.Expression of Pleurocidin from winter flounder in Escherichia coli and optimization of culture conditions.
Xuejiao XU ; Xiangdong ZHA ; Yuanyuan CHE ; Lijuan MA ; Siqun WU ; Peilong YANG ; Huoqing HUANG ; Bin YAO
Chinese Journal of Biotechnology 2016;32(3):365-374
To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.
Animals
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Cloning, Molecular
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Escherichia coli
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metabolism
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Fish Proteins
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biosynthesis
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Flounder
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Recombinant Fusion Proteins
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biosynthesis
3.Construction of novel thioredoxin fusion protein expression system and the production of recombinant Lf-CATH2.
Yiling LU ; Jiuxiang GAO ; Xue QIAO ; Yipeng WANG ; Haining YU
Chinese Journal of Biotechnology 2015;31(3):403-410
The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-pET32α(-TS) was successfully constructed by firstly deleting the thrombin site and S tag from the pET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.
Cathelicidins
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biosynthesis
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Chromatography, Affinity
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Escherichia coli
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Genetic Vectors
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Recombinant Fusion Proteins
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biosynthesis
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Thioredoxins
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genetics
4.Fusion expression of antimicrobial peptides in Escherichia coli.
Xuemin ZHANG ; Lili JIN ; Zheng WANG ; Qiuyu WANG
Chinese Journal of Biotechnology 2014;30(8):1172-1181
Due to their potential application as novel antibiotics, antimicrobial peptides are attracting much attention. Large quantities of highly purified peptides are crucial to basic and clinical studies. Natural resources of antimicrobial peptides are limited and hard to purify, chemical synthesis is of high-cost and unstable, so recombinant expression of antimicrobial peptides is a cost-effective way. Escherichia coli has been the most widely used as host to express antimicrobial peptides with fusion protein, which can not only avoid the lethal effect towards the host, but also protect the peptide from degradation by proteases. Combined with our research, the present article reviews the progress of fusion vector, cleavage methods and optimization options for antimicrobial peptides production with fusion protein in Escherichia coli.
Antimicrobial Cationic Peptides
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biosynthesis
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Escherichia coli
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metabolism
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Genetic Vectors
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Recombinant Fusion Proteins
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biosynthesis
5.Expression and purification of heptad repeat region of the mumps virus F protein and analysis of characteristics.
Yue-Yong LIU ; Ming-Guang FENG ; Jie-Qing ZHU ; Li-Jie JIANG ; Po TIEN
Chinese Journal of Biotechnology 2004;20(3):377-381
Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.
Membrane Fusion
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genetics
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Mumps virus
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genetics
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Recombinant Fusion Proteins
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biosynthesis
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chemistry
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genetics
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isolation & purification
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Repetitive Sequences, Amino Acid
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Viral Fusion Proteins
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biosynthesis
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genetics
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isolation & purification
6.Expression and purification of IFNbeta-HSA fusion protein in Pichia pastoris.
Qi ZHANG ; Jianyong LEI ; Yuedi DING ; Yun CHEN ; Lin QU ; Shuxian CHEN ; Jian JIN
Chinese Journal of Biotechnology 2009;25(11):1746-1752
In order to obtain enough fusion protein for developing preclinical studies of IFNbeta-HAS, we screened Pichia pastoris transformants expressing high-level protein by immunology method. The yield of IFNbeta-HSA was about 500 mg/L by fed-batch fermentation. The purity of IFNbeta-HSA reached 96% through the steps of ultrafiltration, Blue Sepharose FF, Ni2+-IMAC and DEAE Sepharose FF. Analysis of Western blotting showed that IFNbeta-HSA had the antigenicity of IFNbeta and HSA. The specific activity was about 1.96 x 10(7) IU/mg by standard survival activity test on WISH cells challenged with VSV virus. This study provided a method to produce IFNbeta-HSA.
Fermentation
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Humans
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Interferon-beta
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biosynthesis
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genetics
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Pichia
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genetics
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metabolism
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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isolation & purification
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Serum Albumin
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biosynthesis
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genetics
7.Enhancement of functional expression of wheat peroxidase WP1 in prokaryotic system by co-transforming with hemA and hemL of Esherichia coli.
Chao ZHANG ; Liwei SHAN ; Shuaikun SU ; Yanni NAN ; Zhongyu GUO ; Sanhong FAN
Chinese Journal of Biotechnology 2012;28(7):865-876
Wheat grain peroxidase 1 (WP1) belonged to class III plant peroxidase with cofactor heme, which not only has antifungal activity, but also influences the processing quality of flour. In order to enhance functional expression of WP1 in prokaryotic system by increasing endogenous heme synthesis, we constructed a recombinant plasmid pACYC-A-L containing hemA and hemL of Esherichia coli. Then, we co-transformed it into host strain T7 Express with secretive expression vector (pMAL-p4x-WP1) or non-secretive expression vector (pET21a-MBP-WP1), respectively. The MBP-WP1 fusion protein was further purified by amylose affinity chromatography and its peroxidase activity was assayed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate. At 12 h after induction at 28 degree, the extracellular 5-aminolevulinic acid (5-ALA) production of T7 Express/pACYC-A-L was up to 146.73 mg/L, simultaneously the extracellular porphrins also increased dramatically. The peroxidase activity of functional MBP-WP1 obtained from T7 Express/ (pACYC-A-L + pMAL-p4x-WP1) was 14.6-folds of that purified from T7 Express/ pET21a-MBP-WP1. This study not only successfully enhanced functional expression of wheat peroxidase 1 in Esherichia coli, but also provided beneficial references for other important proteins with cofactor heme.
Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Heme
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biosynthesis
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genetics
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Peroxidases
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biosynthesis
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genetics
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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Transformation, Genetic
8.Expression in Pichia pastoris and properties of human serum albumin-interferon alpha2b chimera.
Shao-Hong CHANG ; Xin GONG ; Zhi-Yu YANG ; Tong-Ying WANG ; Guo-Chang MA ; Qing-Jun MA ; Jun WU
Chinese Journal of Biotechnology 2006;22(2):173-179
To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.
Animals
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Bioreactors
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microbiology
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Fermentation
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Humans
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Interferon-alpha
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biosynthesis
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genetics
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Macaca fascicularis
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Pichia
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genetics
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metabolism
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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pharmacokinetics
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Recombinant Proteins
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Serum Albumin
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biosynthesis
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genetics
9.Expression of multi-copy and immuno-reactivity of recombinant type-specific epitope of herpes simplex virus type 1.
Xiao-wei JI ; Xu-hu MAO ; Quan-ming ZOU ; Qing-tan YU ; Li-li ZHAO
National Journal of Andrology 2006;12(7):579-582
OBJECTIVETo express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity.
METHODSMulti-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting.
RESULTSThe recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2.
CONCLUSIONThe HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.
Antigen-Antibody Reactions ; DNA, Recombinant ; Epitopes ; biosynthesis ; immunology ; Herpesvirus 1, Human ; immunology ; Herpesvirus 2, Human ; immunology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Viral Envelope Proteins ; biosynthesis ; immunology
10.Cloning and expression of HSV-I, II type-common antigen gD in Escherichia coli.
Min LI ; Xiaomian LI ; Min LIU
Chinese Journal of Experimental and Clinical Virology 2002;16(2):176-178
BACKGROUNDTo clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD.
METHODSThe authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA.
RESULTSThe constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm.
CONCLUSIONSThe authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics