• Journal of Microbiology and Biotechnology
• /
• 제17권5호
• /
• pp.728-732
• /
• 2007

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT(GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A(MT2A). There are in-framed multiple cloning sites(MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame(ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatography, known as $Ni^{2+}$-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.

• 미생물학회지
• /
• 제44권3호
• /
• pp.178-185
• /
• 2008

조류 독감바이러스(avian influenza virus, AIV)는 사람에게서 발생하는 인플루엔자 대유행에 중요한 역할을 한다. 특히 최근 AIV H9N2형에 의한 가금류 감염이 빈번히 나타나고 있어 인체 감염이 상당히 우려되는 실정이다. 본 연구에서는 최종적으로 AIV의 HA와 NA 단백질에 특이적으로 반응하는 항혈청을 생산하고자 하였다. 먼저 감염된 닭에서 분리된 AIV H9N2 한국분리주 A/Ck/Kr/MS96/96의 게놈RNA로부터 RT-PCR 방법으로 HA와 NA 단백질 N-말단부위에 해당하는 염기서열을 증폭하였다. 이렇게 증폭된 DNA단편은 E. coli 발현벡터 pGEX4T-1에 삽입한 후, BL21 세포에서 각각의 GST fusion protein (GST-HAln와 GST-NAn) 형태로 발현하였다. GST-HAln와 GST-NAn은 모두 glutathione sepharose column을 사용하여 분리 및 정제하였으며, 정제된 단배질을 항원으로 사용하여 토끼 항혈청을 생산하였다. 생산된 항혈청의 항원특이성은 AIV H9N2 한국분리주 A/Ck/Kr/MS96/96로 감염된 MDCK 세포의 cell extract를 사용하여 immunoblotting을 수행함으로써 확인하였다. 본 실험결과AIV H9N2의 HA와 NA단백질 N-말단부위에 해당하는 재조합GST fusion protein과, 이들 각각의 단백질에 특이적으로 반응하는 항혈청은 앞으로AIV 감염의 진단 뿐만 아니라, AIV에 대한 기초연구에 중요한 재료로 사용될 것으로 기대한다.

• BMB Reports
• /
• 제30권5호
• /
• pp.303-307
• /
• 1997

Grb2, which is composed of a Src homology 2 (SH2) domain and two Src homology 3 (SH3) domains, is known to serve as an adaptor protein in signaling for Ras activation. Thus, a blocker of the Grb2 interactions with other proteins can be a potential candidate for an anticancer drug. In this study, we have developed a high throughput screening method for SH3 domain binding ligands and blockers. Firstly, we made and purified the glutathione S-transferase (GST)-fusion proteins with the Grb2 SH2 and SH3 domains, and the entire Grb2. This method measures the binding of a biotin-labeled oligopeptide, derived from a Grb2/SH3 binding motif in the hSos, to the GST-fusion proteins, which are precoated as glutathione S-transferase fusion protein on a solid phase. When $1\;{\mu}g$ of each fusion protein was used to coat the wells, both N- and C- terminal SH3 the domains as well as the whole of Grb2 were able to interact with the biotin-conjugated ligand peptide, while the SH2 domain and GST alone showed no binding affinity. Although N- and C- terminal SH3 domains showed an increase of binding to the ligand peptide in proportion to the amount of peptide, the GST fusion protein with Grb2 demonstrated much higher binding affinity. GST-Grb2 coating on the solid phase showed a saturation curve; 66 and 84% of the maximal binding was observed at 100 and 300 ng/$100\;{\mu}l$, respectively. This binding assay system was peptide sequence-specific, showing a dose-dependent inhibition with the unlabeled peptide of SH3 binding motif. Several other peptides, such as SH2 domain binding motifs and PTB domain binding motif, were ineffective to inhibit the binding to the biotin-conjugated ligand peptide. These results suggest that our method may be useful to screen for new anticancer drug candidates which can block the signaling pathways mediated by SH3 domain binding.

• 한국동물위생학회지
• /
• 제44권4호
• /
• pp.185-194
• /
• 2021

Polyglutamine extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyglutamine aggregates in Huntington's disease (HD). Mutant huntingtin can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. To better understand the mechanism by which an elongated polyglutamine causes aggregates, we have developed an in vitro binding assay system of polyglutamine tract from truncated huntingtin. We made GST-HD exon1 fusion proteins which have expanded polyglutamine epitopes (e.g., 17, 23, 32, 46, 60, 78, 81, and 94 CAG repeats). In the present emergence of new study adjusted nanotechnology on protein chip such as surface plasmon resonance strategy which used to determine the substance which protein binds in drug discovery platform is worth to understand better neurodegenerative diseases (i.e., Alzheimer disease, Parkinson disease and Huntington disease) and its pathogenesis along with development of therapeutic measures. Hence, we used strengths of surface plasmon resonance (SPR) technology which is enabled to examine binding specificity and explore targeted molecular epitope using its electron charged wave pattern in HD pathogenesis utilize conjugated mutant epitope of HD protein and its interaction whether wild type GST-HD interacts with mutant GST-HD with maximum binding affinity at pH 6.85. We found that the maximum binding affinity of GST-HD17 with GST-HD81 was higher than the binding affinities of GST-HD17 with other mutant GST-HD constructs. Furthermore, our finding illustrated that the mutant form of GST-HD60 showed a stronger binding to GST-HD23 or GST-HD17 than GST-HD60 or GST-HD81. These results indicate that the binding affinity of mutant huntingtin does not correlate with the length of polyglutamine. It suggests that the aggregation of an expanded polyglutamine might have easily occurred in the presence of wild type form of huntingtin.

• Animal cells and systems
• /
• 제15권1호
• /
• pp.29-36
• /
• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• Parasites, Hosts and Diseases
• /
• 제54권2호
• /
• pp.239-241
• /
• 2016

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
• /
• pp.619-622
• /
• 2000

For the production of $B^{30}-homoserine$ human insulin precursor, four types of fusion peptides LacZ, MBP, GST, and His-tagged sequence were studied in this work. Recombinant E. coli JM 103 and E. coli JM 109 containing fusion peptides were cultivated at $37^{\circ}C$ for 1hr, and gene expression was occurred when 0.5mM of isopropyl-D-thiogalactoside(IPTG) was added to the culture broth, and followed by longer than 4hr fermentation respectively. DEAE-Sphacel and gel filtration chromatography, amylose and glutathione-Sepharose 4B affinity chromatography, and nickel-affinity chromatography system were employed as purification of $B^{30}-homoserine$ human insulin precursor. Recovery yields of His-tagged, LacZ, GST, and MBP fused $B^{30}-homoserine$ human insulin precursor resulted in 47%, 20%, 20%, and 18%, respectively.

• Biomolecules & Therapeutics
• /
• 제8권2호
• /
• pp.153-159
• /
• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• 대한의생명과학회지
• /
• 제2권1호
• /
• pp.21-30
• /
• 1996

Mitogen-activated protein (MAP) kinase는 여러 세포증식 촉진인자들에 의하여 자신이 인산화됨으로써 활성화되어 다른 protein kinase를 인산화시키는 역할을 하는 세포내 신호전달의 중요한 효소이다. 본 연구에서는 P388 murine leukemia 세포 파쇄액에서 SP sephadex C-50, phenyl superose, Mono Q column을 통하여 MAP kinase를 분리한 결과, 44 kD와 66kD의 isoform을 확인할 수 있었다. 면역 T 세포의 $p56^{kk}$의 N-terminal로부터 유전자 재조합 방법을 통하여 glutathion-s-transferase(GST) fusion protein을 얻은 후 분리한 MAP kinase의 기질로 사용하여 본 결과, wild type과 mutant간에 인산화 정도의 차이를 확인할 수 있어 MAP kinase의 또 다른 기질로 이용할 수 있는 가능성을 제시하였다.

• Journal of Microbiology
• /
• 제34권4호
• /
• pp.311-315
• /
• 1996

The retroviral Gag polyprotein directs the assembly of virion particles and plays an important role in some events after entry into a host cell. The Gag polyprotein of a virus mixture is responsible for inducing murine acquired immunodeficiency syndrome (MAIDS) when injected into susceptible strains of mice. In order to identify the host cellular proteins which interact with the MAIDS virus Gag proteins and possibly mediate the function of the Gag proteins, mouse T-cell leukemic cDNA expression library was screened using the yeast GAL4 two hybrid system. Of 11 individual positive clones, the clone Y1 was selected for the study of protein-protein interaction. Its DNA sequence revealed that it was an exact match to the murine SH3 domain-containing protein SH3P8. It is expressed as 2.4 kbp transcripts in testis at higher levels and in various tissues tested at lower levels. Glutathione S-transferase-Y1 fusion protein binds tightly to $Pr60^{def-gag}$ as well as $Pr65^{eco-gag}$.