• Bulletin of the Korean Chemical Society
• /
• v.29 no.2
• /
• pp.381-388
• /
• 2008

An X-linked skeletal disorder, SEDT (spondyloepiphyseal dysplasia tarda) is a genetic disease characterized by a disproportionately short trunk and short stature caused by mutations in the SEDL gene. This gene is evolutionarily conserved from yeast to human. The yeast SEDL protein ortholog, Trs20p, has been isolated as a member of a large multi-protein complex called the transport protein particle (TRAPP), which is involved in endoplasmic reticulum (ER)-to-Golgi transport. The interaction between SEDL and partner proteins is important in order to understand the molecular mechanism of SEDL functions. We isolated several SEDL-binding proteins derived from rat cells by an immobilized GST-fusion method. Furthermore, the SEDL-homologue proteins were identified using motif based methods. Common motifs between SEDL-binding proteins and SEDL-homologue proteins were classified into seven types and 78 common motifs were revealed. Sequence similarities were contracted to seven types using phylogenetic trees. In general, types I-III and VI were classified as having the function of acetyl-CoA carboxylase, glycogen phosphorylase, isocitrate dehydrogenase, and enolase, respectively, and type IV was found to be functionally related to the GST protein. Types V and VII were found to contribute to TRAPP vesicle trafficking.

• KSBB Journal
• /
• v.15 no.1
• /
• pp.42-48
• /
• 2000

Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

• KSBB Journal
• /
• v.16 no.5
• /
• pp.500-504
• /
• 2001

We scaled up a covalent immobilization system of urokinase to the activated Sepharose and used it repeatedly to cleava a fusion protein consisting of human growth hormone and GST fragment. After scale up from 6 ml to 250 ml. the column system still demonstrated basically the same performance in terms of urokinase immobilization and fusion protein cleavage. When the column was washed with 6 M guanidine HCI after the cleavage reaction, the immobilized urokinase showed no activity probably becasue it was fully unfoled. However, as the denaturant was gradually removed from the column the immobilized urokinase fully regained its bioactivity, which indicated it was properly refolded into is natie conformation as covalently attached to the solid matrix. After 20 cycles of this solid-phase unfolding/refolding. the immobilized urokinase maintained approx. 80% of the initial bioactivity. This method provides and efficient protocol to apply the solid-phase refolding technique to improve the longevity of immobilized enzyme columns.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.85-88
• /
• 2001

We scaled up a covalent immobilization system of urokinase to the activated Sepharose and used it repeatedly to cleave a fusion protein consisting of human growth hormone and GST fragment. After scale up from 6 ml to 250 ml, the column system still demonstrated basically the same performance in terms of urokinase immobilization and fusion protein cleavage. When the column was washed with 6M guanidine HCl after the cleavage reaction. the immobilized urokinase showed no activity probably because it was fully unfolded. However. as the denaturant was gradually removed from the column the immobilized urokinase fully regained its bioactivity. which indicated it was properly refolded into its native conformation as covalently attached to the solid matrix. After 20 cycles of this 'solid-phase unfolding/refolding', the immobilized urokinase maintained approx. 80% of the initial bioactivity. This method provides an efficient protocol to apply the solid-phase refolding technique to improve the longevity of immobilized enzyme columns.

• Journal of Life Science
• /
• v.25 no.4
• /
• pp.385-389
• /
• 2015

In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3. Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein (RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa), were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained from this study indicate that a multi-protein can be stoichiometrically produced from a large poly-protein by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.6 no.2
• /
• pp.144-149
• /
• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• BMB Reports
• /
• v.34 no.6
• /
• pp.537-543
• /
• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1557-1564
• /
• 2009

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.