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 express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.

METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.

RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.

CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.

Carrier Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Humans ; Male ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

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

OBJECTIVE: Construct a recombinant plasmid pET28a-EDA-EDB, prepare the fusion EDA-EDB protein. METHODS: For the production of recombinant fibronectin EDA-EDB in Escherichia coli, the EDA and EDB segments were separated from pGEM2-EDA/EDB and recomposed with two additional amino acids, then cloned into the expression vector pET28a. pET system to express EDA-EDB fusion protein and 6 x His/Ni-NTA system to purify it in a single step were used. Western blotting confirmed the purified protein. RESULTS: The EDA and EDB segments were ligated and inserted into pET28a vector. EDA-EDB fusion protein was highly expressed in Escherichia coli BL21 (DE3). Afterwards, it was purified by Ni-NTA resin and verified by western blotting. CONCLUSION EDA-EDB fusion protein can be expressed in pET system and purified by 6 x His/Ni-NTA system.
Blotting, Western ; Cloning, Molecular ; Escherichia coli/metabolism* ; Fibronectins/biosynthesis* ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins/isolation & purification* ; Recombinant Proteins/isolation & purification*

OBJECTIVETo purify recombinant nuclear protein of Hantaan virus.

METHODSThe recombinant plasmid was transformed into E.coli and induced by IPTG. The expressed protein is a fusion protein with GST and existed in inclusion bodies. The inclusion bodies were processed through denaturation and renaturation and were purified with Glutathione Sepharose 4B affinity chromatography column. Then the purified fusion protein and nuclear protein were examined by sandwich ELISA and Western blot.

RESULTSThe expressed fusion protein was separated from the mixture proteins by the first affinity chromatography. GST was cut from the purified fusion protein with thrombin. The nuclear protein was separated from GST by the second affinity chromatography. The crossed peak represents nuclear protein and the elute peak represents GST. The purified fusion protein and nuclear protein were single band by SDS-PAGE. Both of them had available antigen activity.

CONCLUSIONSPurification of nuclear protein of Hantaan virus with Glutathione Sepharose 4B affinity chromatography is an effective method.

Blotting, Western ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Hantaan virus ; Nuclear Proteins ; analysis ; isolation & purification ; Plasmids ; Recombinant Fusion Proteins ; analysis ; isolation & purification ; Viral Proteins ; analysis ; isolation & purification

Engineering E. coli strain, BL21 (DE3)/pGEX-4T-human Thymosin alpha 1, was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of fusion protein of GST-Thymosin alpha 1 expressed from BL21 (DE3)/pGEX-4T-thymosin alpha 1 is about 35%-40% of total protein after fermentation. Following the simple cut of thrombin or CNBr, about 0.2 g/L thymosin alpha 1 can be harvested. The product is checked by MS and activity test, which indicates that the recombinant product has full biological activity of native thymosin alpha 1.
Escherichia coli ; genetics ; Genetic Engineering ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification ; Thymosin ; analogs & derivatives ; biosynthesis ; genetics ; isolation & purification

Metallothioneins (MT) are potential candidates for medicine development and application. For the purpose of expressing recombinant MT in E. coli, a crab MT cDNA cloned into pGEM-T was subcloned into pET-GST and then transformed into Escherichia Coli BL21. The fusion protein was proved to be expressed in both soluble and insoluble form by SDS-PAGE and western blot. Since metallothionein chelate metal ions, which may effects the physiological process of E. coli, caused the production of recombinant protein was lower than expected. Optimization of the ions content in the culture medium improved expression. The protein was purified by Zn2+ affinity chromatography, and rinsed off with high imidazole (1.5 M) which was the result of MT chelating instead of His-tag. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.
Animals ; Brachyura ; genetics ; Escherichia coli ; genetics ; metabolism ; Metallothionein ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

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 ; chemistry ; Glutathione Transferase ; chemistry ; isolation & purification ; Magnetite Nanoparticles ; chemistry ; Protein Binding ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.
Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; isolation & purification ; Interleukin-6 ; chemistry ; isolation & purification ; Peptide Fragments ; chemistry ; isolation & purification ; Protein Folding ; Recombinant Fusion Proteins ; chemistry ; isolation & purification

OBJECTIVETo establish the expression and purification route for human amelogenin mature peptide in Escherichia coli and obtain the purified amelogenin (AMG) mature peptide.

METHODSRecombined plasmid pGEX-4T-1-AMG was transformed to Escherichia coli BL21. After expression, AMG was purified with glutathione S-transferase fusion protein purification system (GSTrapFF) column.

RESULTSSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization results showed that 45,000 GST-AMG fusing protein and 19,000 target AMG mature peptide were obtained successfully.

CONCLUSIONSpGEX-4T-1-AMG-BL21 system is used successfully to express and purify human AMG mature peptide.

Amelogenin ; biosynthesis ; genetics ; isolation & purification ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Glutathione Transferase ; genetics ; isolation & purification ; Humans ; Peptides ; genetics ; isolation & purification ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism