• 대한의생명과학회지
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• 제18권2호
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• pp.96-103
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• 2012

Kv 4.2 ion channel protein has an ability to open at subthreshold membrane potentials and to recover quickly from inactivation. That is very important for neuronal signal transmission in vertebrate brain. In order to purify Kv 4.2 protein, the novel purification methods were experimented. The purification procedure utilized chromatography on DE-52 ion exchange column and affinity chromatography on a WGA-Sepharose 4B, and Kv 4.2 affinity column chromatography. It was found that 0.5% (wt./vol.) Triton X-100 detergent in lysis buffer worked well for Kv 4.2 protein solubilization from rat brain membrane. Protein quantitative determination was conducted by BCA method at 562 nm for each purification step to avoid determination interference of protein at 280 nm by detergent. The confirmation of Kv 4.2 existence and amount is performed using by SDS-PAGE/immunoblotting or 96-well dot blotting. The Kv 4.2 without interacting protein that contains carbohydrate, was purified from novel biochemical 3-steps purification method for further research.

• BMB Reports
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• 제43권5호
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• pp.319-324
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• 2010

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.

• BMB Reports
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• 제33권2호
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• pp.188-192
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• 2000

Recombinant protein G has been utilized in the purification of antibodies from various mammalian species based on the interaction of antibodies with protein G. The interaction between immunoglobulin and protein G may not be restricted to the Fc protion of antibodies, as many different $F(ab)_2$ or Fab fragments can also bind to protein G. I found both FAb $F(ab)_2$ of 145-2C11, a hamster anti-mouse $CD3{\varepsilon}$ antibody, bound to the protein G-sepharose. Interestingly, Fab and $F(ab)_2$ of 145-2C11 did not bind to the protein A-sepharose. The binding of Fab and $F(ab)_2$ of 145-2C11 to protein G provided a useful method to remove proteases, chopped fragments of the Fc region, and other contaminating proteins. The remaining intact antibody in the protease reaction mixture can be removed by using a protein A-sepharose, because the Fab and $F(ab)_2$ portions of 145-2C11 did not bind to protein A-sepharose. The specific binding of Fab and $F(ab)_2$ portions of 145-sC11 to a protein G-sepharose (though not to a protein A-sepharose) and binding of intact 145-2C11 to both protein A- and G-sepharose will be useful in developing an effective purification protocol for Fab and $F(ab)_2$ portions of 145-2C11.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.550-553
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• 2003

$Hisx_6-GFPuv-Cyt$ c fusion protein을 E. coli 균주 JM109과 BL21 각각에서 발현시킨 결과 발현온도는 $30^{\circ}C$보다 $37^{\circ}C$에서, BL21보다 JM109에서의 발현량이 더 많았다. 그러나 JM109, $37^{\circ}C$에서 발현시킨 fusion protein의 $Ni^{2+}-IDA-agarose$ purification결과 약 45kDa 부근의 fusion protein의 density가 감소되었음을 SDS-PAGE analysis을 통해 알 수 있었다. 또한 western blotting analysis를 통해 이 impurity가 degraded fusion임을 확인 할 수 있었다. degraded fusion은 BL21 균주에서 발현시킨 fusion protein에서도 생성됨을 확인하였다. 모든 결과를 종합해 볼때 $Hisx_6-GFPuv-Cyt$ c fusion protein의 발현은 IM109, $37^{\circ}C$에서 더 많았지만, BL21, $37^{\circ}C$에서 expression시킨 fusion protein이 보다 안정하다고 판단 되어진다. 향후 fusion protein이 bioelectronic device에 적용되려면 degraded fusion protein의 생성을 줄여 activity를 유지하도록 안정한 형태로 발현되어 순수하게 분리정제 되어야 하겠다.

• BMB Reports
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• 제41권5호
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• pp.404-407
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• 2008

A 345-bp gene that encodes human bone morphogenetic protein-2 (hBMP-2) has been synthesized. The codon usage of the resulting gene was modified to include those triplets that are utilized in highly expressed Escherichia coli genes. The hBMP-2 gene was efficiently expressed in E. coli as a soluble and active protein. Since the recombinant hBMP-2 was readily solublized, no further solublization steps were required throughout purification. No additional tagging residues were introduced into the synthetic hBMP-2 gene product. The developed synthetic gene is a promising approach for scaling-up the soluble expression of hBMP-2.

• BMB Reports
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• 제30권3호
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• pp.222-228
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• 1997

Protein phosphatase 2C (PP2C) is one of the four major serine/threonine phosphatases which is dependent on $Mg^{2+}$ for its activity. PP2C was purified from rat liver cytosol and its characteristics were investigated. The substrate employed for routine assay was $[^{32}P]casein$ phosphorylated by PKA. The purification process involved DEAE chromatography, ammonium sulfate fractionation, phenyl sepharose chromatography, sephacryl 5-200 gel filtration, and histone agarose chromatography. The SDS-PAGE of PP2C showed one major single protein band at a position corresponding to a molecular mass of 43 kd and the purification fold was 637. The enzyme showed a pH optimum of 8 and $K_M$ value was $1.9\;{\mu}M$. However, when the substrate was changed to $[^{32}P]histone$, the pH optimum was shifted to 7 and $K_M$ value was $2.3\;{\mu}M.\;Mg^{2+}$ was essential to the enzyme activity and okadaic acid did not exert any inhibitory effect on the enzyme. To examine residue in the active site of PP2C effects of some protein-modifying reagents were tested.

• Journal of Microbiology
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• 제43권3호
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• pp.295-300
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• 2005

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with $Ni^{2+}$ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was $1.26\%$ and $5.56\%$, respectively. It was demonstrated that EBA achieved the highest final protein yield of $9.6\%$ with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.

• Journal of Microbiology
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• 제37권4호
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.

• KSBB Journal
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• 제30권6호
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• pp.291-295
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• 2015

The limited productivity of natural shell matrix proteins has hampered the investigation of their biochemical properties and practical applications, although biominerals in nature obtained by organic-inorganic assemblies have attractive mechanical and biological properties. Here, we prepared a vector for the expression of a fusion protein of a shell matrix protein from Pinctada fucata (named as GRP_BA) with the GRGDSP residue. The fusion protein of BA-RGD was simply produced in E. coli and purified through sequential steps including the treatment with $CaCl_2$ and EDTA solution for cell membrane washing, mechanical cell disruption and the application of non-ionic surfactant of Triton X-100 for BA-RGD inclusion body washing. The production yield was approximately 60 mg/L, any other protein band was not observed in SDS-PAGE and it was estimated that above 97% endotoxin was removed compared to the endotoxin level of whole cell. This study showed this simple and easy purification approach could be applied to the purification of BA-RGD fusion protein. It is expected that the protein could be utilized for the preparation of biominerals in practical aspects.

• Bulletin of the Korean Chemical Society
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• 제32권12호
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.