• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1808-1816
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• 2016

As a scaffolding subunit of the PIK3C3/VPS34 complex, Beclin 1 recruits a variety of proteins to class III phosphatidylinositol-3-kinase (VPS34), resulting in the formation of a distinct PIK3C3/VPS34 complex with a specific function. Therefore, the investigation of a number of Beclin 1 domains required for the protein-protein interactions will provide important clues to understand the PIK3C3/VPS34 complex, of which Beclin1-VPS34 interaction is the core unit. In the present study, we have designed a bacterial overexpression system for the Beclin 1 domain corresponding to VPS34 binding (Vps34-BD) and set up the denaturing purification protocol due to the massive aggregation of Vps34-BD in Escherichia coli. The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.

• BMB Reports
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• v.28 no.6
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• pp.504-508
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• 1995

The electrophoretic patterns of plasma membrane surface proteins of normal rat liver cells and rat hepatomas were compared in 10% non-denaturing and 7-15% gradient non-denaturing gel. Chemical carcinogens, 2-Me DAB (2-methyl-4-dimethylaminoazobenzene) and DENA (diethylnitrosamine), were used to induce hepatoma in rats. One protein which disappeared in hepatoma was identified in normal rat liver by non-denaturing gel electrophoresis. Rabbit antisera were raised against this specific protein, and the protein was purified by Sephacryl S-200 column and immunoaffinity chromatography using the purified antibody. The purified protein showed two bands of molecular weights approximately 50 $kD_{\alpha}$ and 52 $kD_{\alpha}$ by SDS-polyacrylamide gel electrophoresis, which reacted specifically with the antibody. However only one band was observed in non-denaturing gel and also in isoelectric focusing with a pI value of 6.6. This study showed the existence of an unique protein on the plasma membrane surface of normal rat liver cells which disappeared in rat hepatomas induced by chemical carcinogens.

• Applied Biological Chemistry
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• v.41 no.7
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• pp.522-526
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• 1998

Ferritin from germinated soybean seeds was purified by ammonium sulfate precipitation (0.55 saturation), ion-exchange chromatography on DEAE-cellulose, gel filtration chromatography on Sephacryl S-300, and HPLC with Bio-Scale Q2 column. SDS-PAGE analysis showed that the purified ferritin is composed of subunit with an apparent M, 21,000. The molecular mass of the native soybean ferritin estimated by gel filtration on Sephacryl S-300 and non-denaturing polyacrylamide gel electrophoresis appeared to be $510{\sim}560\;kDa$. Soybean ferritin contained 833 mol Fe/mol protein, which is 31-fold more iron than pumpkin ferritin and stained positive for iron on non-denaturing gel. Soybean ferritin cross-reacted with anti-soybean rabbit ferritin antiserum.

• BMB Reports
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• v.28 no.4
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• pp.281-289
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• 1995

Homogeneous human deoxycytidine kinase was purified in one step from a variety of spontaneous human leukemic cells (T-ALL, B-ALL, B-CLL, AML, CML), and from cultured T-lymphoblast cells (MOLT-4) using the newly developed affinity medium, $dCp_4$-Sepharose. Starting with an ammonium sulfate fraction, purification was achieved in one step with the kinase being eluted from a column by the end product inhibitor, dCTP. The purified deoxycytidine kinase from T-ALL cells phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. The enzyme purified from T-ALL and B-CLL cells yielded one major band with a molecular weight of 52 kDa determined by SDS-polyacrylamide gel electrophoresis. AML and CML cells yielded one 52 kDa band and an extra band of 30 kDa molecular weight. On the other hand, B-ALL and MOLT-4 cells showed a low molecular weight band of 30 kDa only. However, the electrophoretic mobilities of enzymatic activity in 12% non-denaturing gels were identical for the dCyd kinase from all different kinds of leukemic cell lines, except that the B-ALL, B-CLL, and MOLT-4 cell preparations had an extra minor peak, all at the same position. dAdo and dCyd phosphorylating activities comigrated indicating that these activities are all associated with the same protein. Two new methods, a disk implantation method and a nitrocellulose powder method were used with a small amount of enzyme protein to raise polyclonal antibodies against dCyd kinase purified from T-ALL cells.

• BMB Reports
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• v.44 no.4
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• pp.279-284
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• 2011

The glutathione S-transferase (GST) system is useful for increasing protein solubility and purifying soluble GST fusion proteins. However, purifying half of the GST fusion proteins is still difficult, because they are virtually insoluble under non-denaturing conditions. To optimize a simple and rapid purification condition for GST-pyruvate kinase muscle 2 (GST-PKM2) protein, we used 1% sarkosyl for lysis and a 1 : 200 ratio of sarkosyl to Triton X-100 (S-T) for purification. We purified the GST-PKM2 protein with a high yield, approximately 5 mg/L culture, which was 33 times higher than that prepared using a conventional method. Notably, the GST-high-temperature requirement A2 (GST-HtrA2) protein, used as a model protein for functional activity, fully maintained its proteolytic activity, even when purified under our S-T condition. This method may be useful to apply to other biologically important proteins that become highly insoluble in the prokaryotic expression system.

• Korean Journal of Veterinary Research
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• v.45 no.2
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• pp.185-189
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• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• Journal of Microbiology
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• v.37 no.4
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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.

• Korean journal of applied entomology
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• v.37 no.1
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• pp.59-64
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• 1998

A juvenile hormone binding protein (JHBP) has been isolated from the whole body homogenate of Galleria rnellonella final instar larvae by gel filtration. The isolated protein is homogenous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. The JHBP has a relative molecular weight of 32 k by denaturing gel electrophresis and 28 k by gel filtration. The protein exhibits a dissociation constant of 3.9 x M for JH 111.

• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.248-252
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• 2001

Recombinant Human serum albumin (rHSA) was purified to near homogeneity from H, polymorpha using heat treatment, ultrafiltratipn and Phenyl Sepharose CL-4B and Mono Q column chro - matographies with a recovery yield of 60% The molecular weight of the purified rHSA was estimated to be about 65,000 Da by denaturing SDS-PAGE The N terminal amino acid sequence of the purified HSA determined by Edman degradation was turned out to be Asp- Ala- His- Lys- Ser- Glu- Ala, suggesting that the rHSA expressed in H, polymorpha was efficiently secreted and correctly processed at the cleavage site of secretion signal sequence. The purified human albumin showed the pI value identical to that of authentic human serum albumin.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.