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 ; chemistry ; Escherichia coli ; genetics ; Protein Folding ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

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 ; genetics ; Mumps virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Repetitive Sequences, Amino Acid ; Viral Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

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

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 ; Baculoviridae ; Crystallization ; Crystallography, X-Ray ; Herpesvirus 2, Human ; chemistry ; Insecta ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Viral Fusion Proteins ; biosynthesis ; chemistry ; genetics

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 ; chemistry ; Chelating Agents ; chemistry ; Chromatography, Affinity ; methods ; Epoxy Compounds ; chemistry ; Histidine ; biosynthesis ; chemistry ; genetics ; Polymers ; chemistry ; Recombinant Fusion Proteins ; isolation & purification ; Sepharose ; chemistry

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 ; chemistry ; Enzyme Activation ; drug effects ; Escherichia coli ; genetics ; metabolism ; Histidine ; chemistry ; Phosphoric Monoester Hydrolases ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism

Amino Acid Substitution ; Exocytosis ; HEK293 Cells ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Membrane Proteins ; chemistry ; metabolism ; Microscopy, Confocal ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Sirolimus ; analogs & derivatives ; chemistry ; metabolism ; Tacrolimus Binding Proteins ; chemistry ; genetics ; metabolism

To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Humans ; Leptin ; chemistry ; genetics ; metabolism ; Molecular Weight ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism

The aim of the study is to construct two vectors for efficient expression of soluble recombinant proteins. The first vector was constructed by cloning the HisSUMO fragment into an expression vector pET30a(+) to fuse with the gene of interest (designated as HisSUMO Express). The second vector was constructed in the same way, but with a hydroxylamine cleavage site between HisSUMO and the gene of interest for an economic purpose (designated as HisSUMO Economic). The mouse fibroblast growth factor-21(mFGF-21), which was difficult to express in routine-used expression vectors, was taken as an example to test the vectors. The results showed that the mFGF-21 was expressed at high level in both vectors. The Sumo/mFGF-21 fusion protein accounted for more than 40% of the total bacterial protein. The fusion protein was purified with Ni-TNA column, and the HisSUMO was removed by cleavage of the fusion protein with either hydroxylamine solution or SUMO protease I. The concentration of the purified mFGF-21 mature protein was 54 mg/L and the recovery rate was 6%. The purified proteins derived from either hydroxylamine or SUMO protease I cleavage could stimulate glucose up-take by adipocytes. These results indicated that both HisSUMO Express and HisSUMO Economic were useful expression vectors for efficient expression of soluble recombinant proteins.
Animals ; Fibroblast Growth Factors ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Hydroxylamine ; chemistry ; Mice ; Peptide Hydrolases ; chemistry ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Solubility