1.Recent progress in fusion enzyme design and applications.
Ziliang HUANG ; Chong ZHANG ; Xi WU ; Nan SU ; Xinhui XING
Chinese Journal of Biotechnology 2012;28(4):393-409
Engineering and redesign of enzymes are important to industrial biocatalysis. Fusion enzyme technology, based on fusion protein design, is frequently used in multifunctional enzyme construction and enzyme proximity control. Here, we reviewed the recent progress in molecular design strategy and application studies of fusion enzymes. The concept and features of fusion enzymes were introduced, followed by a systematical summary of the design strategy of fusion enzymes. In particular, the effects of different linker properties on fusion enzymes and their possible mechanisms were discussed. In addition, recent studies on fusion enzyme applications were also discussed. Finally, based on our own studies on fusion enzymes and the current research progress, the key problems in fusion enzyme technology and perspectives of this field were discussed.
Biocatalysis
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Biotechnology
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Enzymes
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chemistry
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Protein Engineering
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Recombinant Fusion Proteins
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chemistry
2.Refolding of the fusion protein of recombinant enterokinase light chain rEKL.
Chinese Journal of Biotechnology 2006;22(5):811-815
The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in inclusion body in E. coli. The recombinant bacteria was fermented to high density, with high expression of the fusion protein. After being washed with 0.5% Triton X-100 and 4mol/L urea, the inclusion body was dissolved in 6mol/L guanidine and 100mmol/L DTP, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03mg/mL of fusion protein until its final concentration reached 0.3mg/mL. The refolded protein was autocleaved and the active EKL molecule was released after adding 2mmol/L CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95%, with a high activity to cleave the recombinant reteplase fusion protein Trx-rPA. The yield of purified rEKL was more than 60mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large-scale in a way such as expressed in the form of fusion proteins.
Enteropeptidase
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chemistry
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Escherichia coli
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genetics
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Protein Folding
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Recombinant Fusion Proteins
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chemistry
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isolation & purification
3.Expression and purification of DNA binding domain of NR4A1.
Ningning YAN ; Jun LI ; Xiaojuan CHEN ; Yongheng CHEN ; Lin CHEN ; Zhuchu CHEN
Journal of Central South University(Medical Sciences) 2015;40(4):345-350
OBJECTIVE:
To express and purify NR4A1-DNA binding domain (DBD) protein of nuclear receptors.
METHODS:
The fusion protein PET28a-NR4A1-DBD was constructed and purified with the nickel affinity chromatography, cation-exchange chromatography and gel filtration chromatography.
RESULTS:
The protein PET28a-NR4A1-DBD was mostly soluable at 24 °C. A total of 2-3 mg/L pure NR4A1 proteins were yielded in bacterial culture and the purity for final fractions of NR4A1-DBD protein were great than 95% by SDS-PAGE analysis.
CONCLUSION
Nickel affinity chromatography is effective to purify protein. The protein purity can be further improved by the following methods including cation-exchange chromatography and gel filtration chromatography.
Electrophoresis, Polyacrylamide Gel
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Nuclear Receptor Subfamily 4, Group A, Member 1
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chemistry
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Recombinant Fusion Proteins
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chemistry
4.Purification of glutathione-S-transferase fusion protein by glutathione coupled magnetic particles.
Jingjing ZHU ; Liu YANG ; Lei YANG ; Chao CHEN ; Yali CUI
Chinese Journal of Biotechnology 2009;25(8):1254-1260
We established a purification system for glutathione-S-transferase (GST) fusion protein using glutathione coupled magnetic particle. Glutathione was coupled covalently to the surface of magnetic particles with isothiocyanate functional groups. Cell lysate, containing the fusion protein, was then incubated with these glutathione coupled magnetic particles at room temperature. Unbound and non-specifically bound proteins were removed by wash steps. Subsequently, the GST-fusion protein was eluted from the magnetic particles by the addition of reduced glutathione. The resulting fusion protein was tested for purity using SDS-PAGE and demonstrated by Western blotting. The concentration of the fusion protein was measured by Bradford method. Both the conditions for incubation and washing were optimized. The results showed that 150 microg glutathione could be bound on 1 mg of particle surface and 10 mg of the glutatione-coupled magnetic particles was suitable for 100 microL lysate, the optimal incubation time for reaction between particles and lysate was 40 min. The magnetic particles could help purify efficiently GST-fusion protein with a yield of around 516 microg fusion protein per 10 mg particles. Magnetic particles can be successfully used in a simple, rapid and reliable method for the purification of GST-fusion proteins.
Glutathione
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chemistry
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Glutathione Transferase
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chemistry
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isolation & purification
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Magnetite Nanoparticles
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chemistry
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Protein Binding
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Recombinant Fusion Proteins
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chemistry
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isolation & purification
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.Preparation of metal chelate affinity chromatographic medium and its application in the purification of 6 x histidine-tagged protein.
Shu-Juan LI ; Yong-Liang SUN ; Dao-Dao HU ; Chao CHEN ; Ya-Li CUI
Chinese Journal of Biotechnology 2007;23(5):941-946
Using Sepharose CL-6B as support, 3-Chloro-1, 2-epoxypropane as activated agent, carboxymethylated aspartate (CM-Asp) as chelating ligand, A chelate affinity chromatographic medium based on Co2+, named Co-CM-Asp-Sepharose, was prepared and used to purify 6 x His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 microL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The purification results using Co-CM-Asp-Sepharose and Ni-NTA-Agarose (product of Qiagen) were compared. The CD155D1 fusion protein was also purified from 5mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 microL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200 microL of lysate, the optimal incubation time of medium and lysate was 30 min, the optimal imidazole concentration in the eluting buffer was 200 mmol/L, and 200 microg of fusion protein was obtained. In a big scale experiment, 4.6 mg of fusion protein was obtained from 5 mL of lysate using 1.5 mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.
Aspartic Acid
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chemistry
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Chelating Agents
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chemistry
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Chromatography, Affinity
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methods
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Epoxy Compounds
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chemistry
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Histidine
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biosynthesis
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chemistry
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genetics
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Polymers
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chemistry
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Recombinant Fusion Proteins
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isolation & purification
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Sepharose
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chemistry
7.Peptide bond scission of staphylococcal enterotoxin C2 and related factors.
Yue-Bin YING ; Hong-Ying SUN ; Ding DING ; Dan-Xi LI ; Qiao XUE ; Shu-Qing CHEN
Journal of Zhejiang University. Medical sciences 2009;38(5):505-510
OBJECTIVETo investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions.
METHODSThe purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE.
RESULTThe cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2).
CONCLUSIONThe recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.
Amino Acid Sequence ; Enterotoxins ; chemistry ; genetics ; Molecular Sequence Data ; Protein Conformation ; Protein Stability ; Recombinant Fusion Proteins ; chemistry ; genetics
8.Chromatography-assisted refolding of a fusion protein containing multiple disulfide bonds.
Weiquan XIE ; Guifeng ZHANG ; Ling GAO ; Yongdong LIU ; Rong YU ; Zhiguo SU
Chinese Journal of Biotechnology 2010;26(8):1157-1164
To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
Chromatography, Ion Exchange
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methods
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Disulfides
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chemistry
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Fibrinolytic Agents
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analysis
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chemistry
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Hirudins
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analysis
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chemistry
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Protein Refolding
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Recombinant Fusion Proteins
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analysis
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chemistry
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Recombinant Proteins
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analysis
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chemistry
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Tissue Plasminogen Activator
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analysis
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chemistry
9.X-ray diffraction analysis of glycoprotein D from herpes simplex virus type 2.
Zhujun CHEN ; Guangwen LU ; Jianxun QI ; Xiang XU ; Na ZHANG ; Jinghua YAN ; Rongfu WANG
Chinese Journal of Biotechnology 2011;27(10):1499-1506
Glycoprotein D (gD) of Herpes simplex virus type 2 (HSV-2) is a key factor mediating the entry of HSV-2 into host cells. In order to explain the mechanism underlying the gD-mediated receptor-binding and viral entry, we performed a structural study on HSV-2 gD. The ectodomain of the gD protein encompassing residues 1 to 285 was expressed by baculovirus-infected insect cells as a secreted soluble protein with a C-terminal hexa-his tag. The protein was then purified by affinity and size-exclusion chromatography. The purified protein was successfully crystallized using the hanging-drop vapor-diffusion at 18 degrees C in a condition consisting of 0.1 mol/L Hepes pH 7.2, 5% (V/V) 2-methyl-2,4-pentanediol (MPD) and 10% PEG 10 000. The crystals diffracted to 1.8 angstroms resolution and belonged to space group P21, with unit-cell parameters alpha = 63.6, b = 55.4, c = 65.3 angstroms, beta = 96.3 degrees.
Animals
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Baculoviridae
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Crystallization
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Crystallography, X-Ray
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Herpesvirus 2, Human
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chemistry
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Insecta
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genetics
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metabolism
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Recombinant Proteins
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biosynthesis
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chemistry
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genetics
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Viral Fusion Proteins
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biosynthesis
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chemistry
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genetics
10.Determination of the catalytic structures of methyl parathion hydrolase.
Xu-Ping WU ; Wei-Dong LIU ; Hui CAO ; Shun-Peng LI ; Zhong-Li CUI
Chinese Journal of Biotechnology 2005;21(6):998-1002
Methyl parathion hydrolase (MPH) is a novel member of organophosphorus hydrolase. In this study, mpd gene was expressed in Escherichia coli DH5alpha with its native promoter. MPH was purified to homogeneity. Results show that metal-chelating compounds cannot inhabit the enzyme activity. Inductively Coupled Plasma-Atomic Emission Spectrometry analysis showed that MPH is a zinc-containing enzyme, the Zinc to enzyme molar ratio is near 2:1. In order to investigate critical residues related to enzymatic activity of MPH, chemical modification reagents EDC, DEPC, butanedione and pyridoxal were tested. Experiment results suggested that aspartate, glutamate, arginine and lysine are not important for enzyme activity. But DEPC, which can modify histidine residue, inactivate the enzyme activity greatly, and the inactivation rate is 9.6 h(-1). This result reflects that histidine residues are essential for enzyme activity. All these results provide basic data for MPH structure and molecular evolution research.
Aryldialkylphosphatase
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chemistry
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Enzyme Activation
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drug effects
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Escherichia coli
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genetics
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metabolism
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Histidine
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chemistry
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Phosphoric Monoester Hydrolases
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chemistry
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Recombinant Fusion Proteins
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biosynthesis
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chemistry
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genetics