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

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

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

Polyelectrolyte with a large number of cations or anions could precipitate the oppositely charged proteins to form polyelectrolyte-protein complexes, which then aggregated to form larger particles via electrostatic attraction or hydrophobic interaction. The precipitation was affected by the molecular weight and concentration of the polyelectrolyte as well as the ionic strength and pH of the solution. The use of precipitation is an efficient method for selective separation of proteins from crude biological mixtures in the downstream processes of bioengineering.
Acrylic Resins ; chemistry ; Chemical Precipitation ; Electrolytes ; chemistry ; Polyethyleneimine ; chemistry ; Protein Binding ; Proteins ; isolation & purification

OBJECTIVETo explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis (2-DE) analysis.

METHODSThe extracts of Dermatophagoides pteronyssinus were prepared with Coca's solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid (BCA) assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis.

RESULTSThe concentrations of extracted protein by methods of Coca's solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight (LMW) range could be detected with the Coca's solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight (MMW) protein spots were absent. Several MMW protein spots (174-178 kD and 133 kD) and more LMW protein spots were detected with Trizol reagent method.

CONCLUSIONSAmong Coca's solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.

Allergens ; isolation & purification ; Animals ; Dermatophagoides pteronyssinus ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; Guanidines ; chemistry ; Phenols ; chemistry ; Proteins ; isolation & purification

Monomethoxy Polyethylene Glycol(mPEG20000) was activated by N-hydroxysuccinimede and analyzed by infrared spectrum and hydrolysis kinetics. In order to propose the optimized reaction conditions of mono-PEGylated rhG-CSF, orthogonal design of the experiment was investigated. Ion exchange chromatography was used to separate and purify PEGylated rhG-CSF from unPEGylated rhG-CSF. The purity of mono-PEGylated rhG-CSF was analyzed by high performance liquid chromatography (HPLC) to be 97%.
Granulocyte Colony-Stimulating Factor ; chemistry ; isolation & purification ; Humans ; Polyethylene Glycols ; chemistry ; Recombinant Proteins ; chemistry ; isolation & purification ; Surface Properties

In this study, polyacrylicacid precipitation alkalescence protein from Momordica charantia L. seeds was studied, and the effect of conditions on experiment was also evaluated. Isoelectric precipitation is achieved by adjusting the pH of a protein solution and is based on that a protein's solubility is at minimum at its pI. The sample was titrated to pH 6.0 with citric acid, and 14.62% proteins were precipitated. With hydrochloric acid to pH 4.0, 32.49% proteins were precipitated. With the acetic acid to pH 6.0 and pH 4.0, 26.17% and 38.72% proteins were precipitated, respectively. In the 1 mL Bitter melon seeds extraction(pH 4.0) adjusted by acetic acid, hydrochloric acid and citric acid, the optimum dosage of PAA (1%) precipiting alkalescency protein (pl 8.65-9.30) was 100 microL, 120 microL and 100 microL, respectively. The respective extraction (1mL) was titrated to pH 5.0, pH 4.0, and pH 3.0 by acetic acid. After isoelectric precipitation, the PAA precipitation protein was performed. When concentration of PAA (1%) was 160 microL/mL, the protein decreased in the supernatant was 33.77% at pH 5.0, and 43.56% at pH 3.0. When concentration of PAA (1%) was 120 microL/mL, the protein decreased in the supernatant was 30.83% at pH 4.0. PAA-Protein complex could redissolve in alkaline conditions (pH > 9.0) and the protein most easilly redissolved when the NaCL was 3.0%. The bitter melon seeds extraction after PAA purification flowed through the Sephadex G-75 columns. The peaks I and II were obtained after 175 min and 300 min, respectively. SDS-PAGE and IEF analysis showed that the molecule weight from peaks I was 30 kD with pI 9.5, peaks II 10 kD with pI 9.3.
Acrylic Resins ; chemistry ; Chemical Precipitation ; Momordica charantia ; chemistry ; Plant Extracts ; chemistry ; Plant Proteins ; chemistry ; isolation & purification ; Seeds ; chemistry

White-rot fungus manganese peroxidase (MnP) that has great potential in degrading azo dyes is one of the extracellular glycolsylated heme proteins. MnP from Schizophyllum sp. F17 was isolated and purified by Sephadex G-75 gel filtration chromatography followed by DEAE-cellulose anion exchange chromatography. The molecular weight of the puried enzyme was 49.2 kDa, while the half-life of the MnP in the presence of 0.1 mmol/L H2O2 was 5-6 min. The efficiency of MnP-catalyzed reactions were determined by three key factors: the concentrations of Mn2+, H2O2, and the amount of MnP. Using single factor analysis, an optimized concentration of Mn2+, H2O2 and enzyme were optimized to be 1.2 mmol/L, 0.1 mmol/L, and 0.4 mL, respectively. A response surface methodology (RSM) employing two-level-three-factor full factorial central composite design was used to optimize the catalytic conditions. The result showed that the concentration of H2O2 and the interaction between H2O2 and MnP mostly affect the MnP catalytic efficiency. Finally, we show that the azo dyes could be efficiently decolorized by the purified MnP under optimized conditions.
Azo Compounds ; chemistry ; isolation & purification ; metabolism ; Catalysis ; Coloring Agents ; chemistry ; isolation & purification ; metabolism ; Environmental Pollutants ; chemistry ; isolation & purification ; metabolism ; Fungal Proteins ; chemistry ; isolation & purification ; metabolism ; Peroxidases ; chemistry ; isolation & purification ; metabolism ; Schizophyllum ; enzymology

OBJECTIVETo establish an efficient and high resolution 2-DE (two-dimensional electrophoresis) protocol for root tuber proteome analysis of Rehmannia glutinosa.

METHODProteins from root tuber of R. glutinosa were extracted by using five different methods and their productivity and profiles were assessed by means of SDS-PAGE and two-dimensional electrophoresis.

RESULTThe trichloroacetic acid (TCA)-phenol extraction method was found most effective for the extraction with the highest protein yield, the most spots in protein patterns, and the highest resolution of proteins, and the clearest background could be achieved simultaneously. A 1:5 solution of ampholine pH 3-10 and pH 5-8 for a nonlinear gel and the 170 microg of protein loading dosage obtained maps with more protein spots and higher resolution of separation patterns.

CONCLUSIONThis study based on the optimized root tuber proteome preparation and the 2-DE protocol gets a high resolution and reproducibility 2-DE image, which will be expected to have excellent applications in proteomics studies of R. glutinosa tuber root.

Electrophoresis, Gel, Two-Dimensional ; methods ; Plant Proteins ; analysis ; chemistry ; Plant Roots ; chemistry ; Proteome ; chemistry ; isolation & purification ; Rehmannia ; chemistry