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

• BMB Reports
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• v.34 no.2
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• pp.150-155
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• 2001

The mosquito-larvicidal Cry4B protein from Bacillus thuringiensis subsp. israelensds was expressed in Escherichia coli. Upon activation by trypsin, the 130-kDa protoxin was processed into the 65-kDa active toxin containing two polypeptide fragments of ca. 47 and ca. 20 kDa. These two polypeptides are products of internal cleavages on the exposed loop connecting helices 5 and 6 in the seven-helical bundle domain. PCR-based mutagenesis was employed to introduce an additional cleavage site into the loop connecting helices 3 and 4. A series of amino acid changes were introduced into the targeted loop, resulting in seven mutant protoxins. Upon digestion with trypsin, a group of mutants with arginine introduced into the loop (EPRNQ, EPNRNQ, EPRNP, ESRNP and SSRNP) produced polypeptide products similar to those of the wild type (EPNNQ). When the loop, SSRNP, was expanded by an insertion of either asparagine (NSSRNP) or valine (VSSRNP), an additional cleavage was detected with proteolytic products of 47,12 and 6 kDa. This cleavage was confirmed to be at the introduced arginine residue by N-terminal sequencing. The mosquito larvicidal assay against Aedes aegypti demonstrated a relatively unchanged toxicity for the mutants without cleavage and reduced toxicity for those with an additional cleavage.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.911-917
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• 2009

The apoptotic caspases have been classified in accordance with their substrate specificities, as the optimal tetrapeptide recognition motifs for a variety of caspases have been determined via positional scanning substrate combinatorial library technology. Here, we focused on two proteolytic recognition motifs, DEVD and IETD, owing to their extensive use in cell death assay. Although DEVE and IETD have been generally considered to be selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, we attempted to monitor the cleavage preference for caspase-3, particularly using the recombinant protein substrates. For this aim, the chimeric GST:DEVD:EGFP and GST:IETD:EGFP proteins were genetically constructed by linking GST and EGFP with the linkers harboring DEVD and IETD. To our best knowledge, this work constitutes the first application for the monitoring of cleavage preference employing the recombinant protein substrates that simultaneously allow for mass and fluorescence analyses. Consequently, GST:IETD:EGFP was cleaved partially in response to caspase-3, whereas GST:DEVD:EGFP was completely proteolyzed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Collectively, using these chimeric protein substrates, we have successfully evaluated the feasibility of the recombinant protein substrate for applicability to the monitoring of cleavage preference for caspase-3.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• BMB Reports
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• v.51 no.5
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• pp.211-218
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• 2018

Chromatin is an intelligent building block that can express either external or internal needs through structural changes. To date, three methods to change chromatin structure and regulate gene expression have been well-documented: histone modification, histone exchange, and ATP-dependent chromatin remodeling. Recently, a growing body of literature has suggested that histone tail cleavage is related to various cellular processes including stem cell differentiation, osteoclast differentiation, granulocyte differentiation, mammary gland differentiation, viral infection, aging, and yeast sporulation. Although the underlying mechanisms suggesting how histone cleavage affects gene expression in view of chromatin structure are only beginning to be understood, it is clear that this process is a novel transcriptional epigenetic mechanism involving chromatin dynamics. In this review, we describe the functional properties of the known histone tail cleavage with its proteolytic enzymes, discuss how histone cleavage impacts gene expression, and present future directions for this area of study.

• BMB Reports
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• v.52 no.4
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• pp.239-249
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• 2019

Membrane-anchored full-length MET stimulated by its ligand HGF/SF induces various biological responses, including survival, growth, and invasion. This panel of responses, referred to invasive growth, is required for embryogenesis and tissue regeneration in adults. On the contrary, MET deregulation is associated with tumorigenesis in many kinds of cancer. In addition to its well-documented ligand-stimulated downstream signaling, the receptor can be cleaved by proteases such as secretases, caspases, and calpains. These cleavages are involved either in MET receptor inactivation or, more interestingly, in generating active fragments that can modify cell fate. For instance, MET fragments can promote cell death or invasion. Given a large number of proteases capable of cleaving MET, this receptor appears as a prototype of proteolytic-cleavage-regulated receptor tyrosine kinase. In this review, we describe and discuss the mechanisms and consequences, both physiological and pathological, of MET proteolytic cleavages.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.39-40
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• 1995

Thymopoietin(TP) was originally isolated from bovine thymic extracts on the basis of its ability to affect neuromuscular transmission when injected into mice (Goldstein, 1974). A 49 amino acid polypeptide was isolated and sequenced (Schlesinger and Goldstein, 1975). It is now evident that this molecule was created by proteolytic cleavage of larger thymopoietin proteins during isolation, and represents the N-terminal sequence of these proteins. Nevertheless, this proteolytic fragment was active in both neurophysiological and immunological experiments, and enabled the identification of an active pentapeptide. (amino acids 32 to 36, Arg-Lys-Asp-Val-Tyr, thymopentin), which. has been studied as an immunomodulatory drug.

• Journal of Life Science
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• v.10 no.2
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• pp.218-227
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• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.

• Restorative Dentistry and Endodontics
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• v.22 no.1
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• pp.76-92
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• 1997

Porpilyromonas endodontalis is specifically involved in endodontic infections. The bacterium can be isolated almost exclusively only from infected rool canals. P. gingivalis also has been implicated in endodontic infection. Pathogemcity of P. gingival is is attributed to a variety of virulence factors, especially proteases, produced by the bacterium. Importance of P. endodontalis in endodontic infection has been revealed. However, the pathogenic property of P. endodontalis has not been extensively studied. The present study was undertaken to characterize the proteolytic activity of P. endodontalis and compare the activity with that of P. gingivalis which has the most potent and diverse proteases among oral bacteria. For this purpose, culture supematants(SUP) and cell extracts(CE) were obtained from these two bacteria and were subjected to zymography using 15% polyacrylamide gel copolymerized with gelatin, type I, IV collagens or albumin. Hydrolysis of the collagens was further investigated by the cleavage assay using native type I and IV collagens in solution-phase. The results were as follows: 1. P. endodontalis apparently has a proteolytic activity that is comparable with that of P. gingivalis. 2. SUP and CE obtained from P. endodontalis and P. gingival is showed the strongest activity for gelatin, followed by type I and IV collagens, and albumin. 3. In the zymography, no noticeable difference in proteolytic activity for gelatin and albumin between the SUP and CE was observed, but in the cleavage assay using native collagens, the SUP showed a stronger collagenolytic activity than the CE. 4. The gelatinolytic activity of both the SUP and CE from these two bacteria was diminished in the presence of $CaCl_2$ or reducing agents such as ${\beta}$-mercaptoethanol and dithiothreitol(DTT). 5. Type I(calf skin and human placenta) collagenolytic activity of P. endodontalis and P. gingivalis was reduced by DTT but not affected by $CaCl_2$. The inhibitory effect of DTT, however, was reduced to some extent by $CaCl_2$. 6. Type IV collagenolytic activity of these two bacteria was not affected by $CaCl_2$ but increased to some extent in association with the reducing agents. 7. Hydrolysis of albumin by P. endodontalis and P. gingivalis was demonstrated only in the presence of the reducing agents. The overall results indicate that with respect to proteolytic activity, P. endodontalis appears to be as potent as P. gingivalis, or maybe more, and its proteolytic characteristic is similar to that of P. gingivalis. This suggests that P. endodontalis has so potent proteolytic activity that can participate by itself in endodontic infections and apical periodontitis, causing tissue destruction.

• The Journal of Korean Society of Virology
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• v.29 no.3
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• pp.183-193
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• 1999

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is synthesized as a 160 KDa precursor, gp160, that is cleaved by a cellular protease to form the gp120 and gp41 subunits. Mammalian expression vectors were designed that are capable of efficient expression of various mutant envelope glycoproteins derived from a molecular clone of HIV-1. To construct these vectors, one type of mutation was made at the gp120-gp41 cleavage site by oligonucleotide-directed mutagenesis. And another mutation was made to change amino acids in the membrane spanning region of HIV-1 gp41 important for membrane anchorage. Next, these two mutations were combined to generate a vector to have double mutations in cleavage site and membrane-spanning region. These mutants were transiently expressed in mammalian cells. The effect of these mutations on envelope glycoprotein synthesis, proteolytic processing and secretion was determined. In addition, cell surface expression and ability of the glycoprotein to induce syncytium formation were examined. This study provides a mammalian expression system that is capable of efficient expression and secretion of soluble gp160.