Fusion tags are originally developed to facilitate the purification of recombinant protein from crude extracts. In recent years, the discovery of different tags and the development of fusion strategy make the function of fusion tags diversified. However, there was no a cure-all fusion tag for different applications. We here give an overview of fusion tag technology and the different applications of fusion tags, including the purification, detection and oriented immobilization of recombinant protein, the visualization of bioevent in vivo, the enhancement of the yield of protein, the improvement of the solubility and stability of the expressed protein.
Recombinant Proteins ; chemistry ; isolation & purification ; Solubility

Human serum albumin(HSA) has been used clinically to treat a number of diseases with high dosage. Extremely pure puoduct is required in large-scale production. Plasma-derived HSA(pHSA) has long been produced by precipitation methods. Among them cold ethanol precipitation is dominant. However, chromatographic purification of HSA has been increasingly studied in the last few years. Application of chromatography, especially ionexchange, affinity, and size-exclusion, has opened a new area in the production of pHSA. A new challenge is the purification of recombinant HSA(rHSA). A successful approach involves STREAMLINE expanded bed adsorption to direct capture the target product from the fermentation broth. This novel process eliminates the need to separate the cells by centrifugation or membrane filtration. Ion exchange chromatography and hydrophobic chromatography play a central role in the purification scheme. Integration with other chromatographic techniques such as size-exclusion, metal chelate, and affinity gives improved purification results. Though innovative, the purification of rHSA still needs further improvement and optimization to increase product purity and process recovery.
Humans ; Recombinant Proteins ; isolation & purification ; Serum Albumin ; isolation & purification

OBJECTIVETo purify the recombinant human cellular repressor of EIA stimulated gene (hCREG)/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro.

METHODSThe recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration (0.5 microg/ml, 1 microg/ml and 2 microg/ml) on proliferation of HITASY cells was studied by flow cytometric analysis and the effect of recombinant protein on proliferation of HITASY cells was confirmed by BrdU incorporation method.

RESULTSThe recombinant hCREG protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/ml and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometric analysis. The proliferation of HITASY cells cultured in vitro with 2 microg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups (P < 0.05).

CONCLUSIONThe purification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.

Adenoviridae ; Cell Division ; Cells, Cultured ; Glycoproteins ; isolation & purification ; Glycosylation ; Humans ; Recombinant Proteins ; isolation & purification ; metabolism ; Repressor Proteins ; isolation & purification ; metabolism

Annexin A5 ; biosynthesis ; isolation & purification ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; isolation & purification

According to the amino acid sequence of des-pGlu1-Brazzein, 4 pairs of oligonucleotide with cosmic site were synthesized by using yeasty biased codons. After linkage and PCR, the 179 bp code area of des-pGlu1-Brazzein was obtained and inserted into pPIC9K, which resulted in the recombinant expression vector pPIC9K-Bra. By digestion with Sal I, the lined pPIC9K-Bra was transformed into Pichia pastoris GS115 by electric shock. The results of expression indicted that the secreted target protein accounted for 51.6% of total protein in the supernatant and showed biological activity after purification.
Codon ; Pichia ; genetics ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sweetening Agents

Protein disulfide isomerase-related protein A (PRPA) was highly expressed (about 34%) in Escherichia coli by inserting the whole PRPA cDNA into the vector pET23b. After expression, the purified protein was acquired through ammonium fractional precipitation and Bio-Rex 70 chromatography. PRPA shows low disulfide isomerase activity (only about 1/250 of that of hPDI), decreases the reactivation yield of denatured and reduced lysozyme either in redox and non-redox Hepes buffer or redox PBS buffer and facilitates the aggregation of denatured and reduced lysozyme. Fluorescence spectra of PRPA indicate that PRPA has more hydrophobic groups at surface than that of hPDI, and which can be used to explain why PRPA has anti-chaperone activity during the refolding of denatured and reduced lysozyme.
Cloning, Molecular ; Fungal Proteins ; chemistry ; genetics ; isolation & purification ; Muramidase ; chemistry ; Plasmids ; Protein Folding ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Spectrometry, Fluorescence

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

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

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