• Journal of Microbiology and Biotechnology
• /
• 제25권2호
• /
• pp.247-254
• /
• 2015

In this presentation, I describe the expression and purification of the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a commercially available double cistronic vector, pACYCDuet-1, to express the receptor heterodimer in a single cell as the soluble form. I describe here the expression and characterization of a biologically active heterodimer composed of the liver X receptor β-ligand binding domain and retinoid X receptor α-ligand binding domain. Although many of these proteins were previously seen to be produced in E. coli as insoluble aggregates or "inclusion bodies", I show here that as a form of heterodimer they can be made in soluble forms that are biologically active. This suggests that co-expression of the liver X receptor β-ligand binding domain with its binding partner improves the solubility of the complex and probably assists in their correct folding, thereby functioning as a type of molecular chaperone.

• Journal of Microbiology and Biotechnology
• /
• 제26권10호
• /
• pp.1701-1707
• /
• 2016

Lipoxygenase (LOX) is an industrial enzyme with wide applications in food and pharmaceutical industries. The available structure information indicates that eukaryotic LOXs consist of N terminus β-barrel and C terminus catalytic domains. However, the latest crystal structure of Pseudomonas aeruginosa LOX shows it is significantly different from those of eukaryotic LOXs, including the N-terminal helix domain. In this paper, the functions of this N-terminal helix domain in the soluble expression and catalysis of P. aeruginosa LOX were analyzed. Genetic truncation of this helix domain resulted in an insoluble P. aeruginosa LOX mutant. The active C-terminal domain was obtained by dispase digestion of the P. aeruginosa LOX derivative containing the genetically introduced dispase recognition sites. This functional C-terminal domain showed raised substrate affinity but reduced catalytic activity and thermostability. Crystal structure analyses demonstrate that the broken polar contacts connecting the two domains and the exposed hydrophobic substrate binding pocket may contribute to the insoluble expression of the C terminus domain and the changes in the enzyme properties. Our data suggest that the N terminus domain of P. aeruginosa LOX is required for its soluble expression in E. coli, which is different from that of the eukaryotic LOXs. Besides this, this N-terminal domain is not necessary for catalysis but shows positive effects on the enzyme properties. The results presented here provide new and valuable information on the functions of the N terminus helix domain of P. aeruginosa LOX and further improvement of its enzyme properties by molecular modification.

• 미생물학회지
• /
• 제37권4호
• /
• pp.245-252
• /
• 2001

MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인 Erm 단백질들은 아미노산 서열 중 그 동일성과 유사성이 높아 구조적으로도 동등한 단백질의 한 집단을 형성한다. 최근 X-ray crystallography에 의해 구조가 결정된 ErmC\` 및 ErmAM 단백질의 구조에 근거하여 ErmSF 단백질도 catalytic domain과 substrate binding domain으로 구분하였고 N-terminal end에 존재하는 catalytic domain의 대량생산을 다양한 pET 발현 vector를 사용하여 시도하였다. 그리고 catalytic domain을 coding하는 DNA 절편은 세 종류를 사용하였다: DNA 절편 1은 Met 1부터 Glu 186까지를 coding하고 DNA 절편 2는 Arg 60부터 Glu 186까지의 정보를 가진 DNA이고 DNA 절편 3은 Arg 60부터 Arg 240까지를 encoding하는 DNA이다. 사용된 다양한 발현 vector중에서 pET19b는 DNA 절편 3, pET23b는 DNA 절편 1과 2를 성공적으로 대량생산하였다. 그러나 대량생산된 catalytic domain들은 불용성 단백질 집합체인 inclusion body를 형성하였다. ErmSF catalytic domain들의 용해성 단백질의 생산을 위하여 chaperone GroESL과 Thioredoxin의 동시 발현 및 배양온도를 $22^{\circ}C$로 낮추어 시도했으나 대량 발현된 단백질의 용해에는 도움을 얻지 못하였다.

• Animal cells and systems
• /
• 제13권4호
• /
• pp.411-418
• /
• 2009

We are reporting the identification, expression patterns, and possible biological functions of zebrafish kif3b (kif3bz) encoding 475 amino acids. Kif3Bz contains the kinesin motor domain, catalytic domain, KISc domain, and one single coiled coil domain. Phylogenetic analysis indicates that kif3bz is a highly conserved gene among the tested vertebrates. First of all, both maternal and zygotic messages of kif3bz were evenly distributed in the blastomeres at 2-cell stage. Its ubiquitous expression throughout the blastomeres continued till 40% epiboly. However, kif3bz transcripts became restricted in Kupffer's vesicle at tailbud and 6-somite stages. At 13-somite stage, kif3bz expression pattern became specific to the telencephalon, diencephalon, trigeminal placode, and somites. Such expression patterns were further intensified in the telencephalon, diencephalons, hind brain, pronephric ducts, optic vesicles, and spinal cord neurons in the 23-somite stage embryos, and last till 24 hpf. We discussed possible functions of Kif3Bz related to the vertebrate embryonic development.

• Bulletin of the Korean Chemical Society
• /
• 제28권9호
• /
• pp.1549-1552
• /
• 2007

The cytoplasmic domain of syndecan-4, a type I transmembrane heparan sulfate proteoglycan, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF and NMR spectroscopy. The yield of the peptide was 3.0-1.5 mg per liter in LB or minimal medium, respectively. The recombinant expression and purification of this domain will enable us its structural and functional studies using multidimensional NMR spectroscopy.

• Bulletin of the Korean Chemical Society
• /
• 제31권9호
• /
• pp.2607-2612
• /
• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

• Journal of Plant Biotechnology
• /
• 제32권4호
• /
• pp.269-273
• /
• 2005

Methylotrophic 효모 Pichia pastoris 발현시스템을 사용하여 인간 TLR9 단백질의 세포내 TIR 도메인을 발현하였다. TIR 단백질이 P. pastoris에서 발현되어 배지 속으로 분비되는 것을 SDS-PAGE로 확인하였고, 발현된 단백질을 western-blot, MALDI-TOF 질량분석으로 동정하였다. 이를 통하여 TIR 딘백질이 P. pastoris에서 안정적으로 발현됨을 알 수 있었다. 그리고 발현된 단백질을 니켈 친화, 양이온교환수지, 겔 투과 크로마토그라피를 사용하여 순수 분리 정제하였다. P. pastoris를 이용한 단백질의 발현과 정제방법은 대장균에서 잘 발현되지 않는 단백질의 발현에 응용될 수 있을 것이다.

• 산업기술연구
• /
• 제36권
• /
• pp.33-37
• /
• 2016

Cellulose binding domains (CBDs) of cellulases are thought to assist in the hydrolysis of insoluble crystalline cellulose. To gain sufficient amount of CBDs, the self-cleavable intein tag was used for expression and purification of Trichoderma reesei cellobiohydrolase I CBD in E. coli. Synthetic CBD genes, CBD or linker-CBD were cloned into expression vector pTYB11. Recombinant CBDs were successfully purified by intein mediated purification with an affinity chitin-binding domain. The final yields of recombinant CBD and linker-CBD were 3.2 mg/L and 1.4 mg/L, respectively. The functional bindings of recombinant CBDs were confirmed by Avicel binding experiments. The simple and easy purification method using self-cleavable intein tag can be further used in pretreatment of crystalline cellulose or characterization of engineered CBDs.

• 한국자기공명학회논문지
• /
• 제14권2호
• /
• pp.105-116
• /
• 2010

SH3YL1, a novel protein containing one Src homology 3 domain at the carboxyl terminus was first detected in mouse anagen skin cDNA. This protein had a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein. The sequence identity was remarkable at the carboxyl and amino-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein and thus was termed as SH3YL1. SH3YL1 is composed of two domains, a DUF500 at N-termini and a SH3 domain at C-termini. In our study we cloned the SH3 domain in bacterial expression system in Escherichia coli using pET32a vector with TEV protease cleavage site and purified as a monomer using affinity chromatography. The N-terminal poly-Histidine tag was cleaved with TEV protease and target protein was used for backbone studies. Our study showed that SH3 domain primarily consists of $\beta$-sheet which is in consistence with previous result performed on the truncated SH3 domain of SH3YL1.

• JSTS:Journal of Semiconductor Technology and Science
• /
• 제9권1호
• /
• pp.8-13
• /
• 2009

In this paper, discontinuous interconnect lines are modeled as a cascaded line composed of many uniform interconnect lines. The system functions of respective uniform interconnect lines are determined, followed by its time domain response. Since the time domain response expression is a transcendental form, the waveform expression is reconfigured as an approximated linear expression. The proposed model has less than 2% error in the delay estimation.