• YAKHAK HOEJI
• /
• v.52 no.6
• /
• pp.466-470
• /
• 2008

Human cytomegalovirus expresses an unusual protein kinase UL97, a member of ${H_V}{U_L}$ family of protein kinase. UL97 can phosphorylate nucleoside analogs such as ganciclovir as well as protein/peptide. It has previously been reported that UL97 is able to phosphorylate a KESYSVYVYKV peptide and that P+5 position (K) is important. We examined the extent of contribution of other positions (P-4 through P+6) of the peptide to be substrate of UL97 using alanine substituted peptides (Ala scanning) and deleted peptides. The result suggested that the E (P-2) is negative effect and P+5 (K) is still important. The peptide YSVYVYK is the shortest substrate enough to show high activity, which could be a starting point to develop peptidomimetic drug. This study would give important information to deeply understand the substrate specificity of UL97 and develop an antiviral drug using the small peptide identified here.

• Microbiology and Biotechnology Letters
• /
• v.24 no.1
• /
• pp.67-71
• /
• 1996

The plasmid pK8 was constructed to verify the existence of an adenylate domain in peptide synthetase by using pGC12. 1.2 kb fragment, coding tyrocidine synthetase 1 (123 kDa) was deleted, and 79.6 kDa one was expressed in Escherichia coli XL1-blue. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over expressed synthetase. ATP-[$^{32}P$]PPi exchange reaction was measured for the enzyme assay.

• BMB Reports
• /
• v.32 no.6
• /
• pp.561-566
• /
• 1999

CA(1-8)-ME(1-12) [CA-ME], composed of cecropin A(1-8) and melittin(1-12), is a synthetic antimicrobial peptide having potent antibacterial and antitumor activities with minimal hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly, of CA-ME on antibiotic activity, CA-ME and three analogues, CA-ME1, CA-ME2, and CA-ME3, were synthesized. The Gly-Ile-Gly sequence of Ca-ME was deleted in CA-ME1 and replaced with Pro and Gly-Pro-Gly in CA-ME2 and CA-ME3, respectively. CA-ME1 and CA-ME3 showed a significant decrease in antitumor activity and phospholipid vesicle-disrupting ability. However, CA-ME2 showed similar antitumor and vesicle-disrupting activities, as compared with CA-ME. These results suggest that the flexibility or ${\beta}$-turn induced by Gly-Ile-Gly or Pro in the central part of CA-ME may be important in the electrostatic interaction of the N-terminus cationic ${\alpha}$-helical region with the cell membrane surface and the hydrophobic interaction of the C-terminus amphipathic ${\alpha}$-helical region with the hydrophobic acyl chains in the cell membrane. CA-ME3 exhibited lower antitumor and vesicle-disrupting activities than CA-ME and CA-ME2. This result suggests that the excessive ${\beta}$-turn structure caused by the Gly-Pro-Gly sequence in CA-ME3 seems to interrupt ion channel/pore formation in the lipid bilayer. We concluded that the appropriate flexibility or bilayer. We concluded that the appropriate flexibility or ${\beta}$-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.1
• /
• pp.113-118
• /
• 1999

The insulin-like growth factor (IGF) is found in all vertebrates and its type-II molecule is regarded as a fundamental embryonic growth factor during development. We have firstly identified, in this study, a cDNA clone corresponding to IGF-II (flIGF-II) from the adult brain of the teleost, Paralichthys olivaceus. We also examined the tissue expression of flIGF-II in several adult tissues by RT-PCR. The flIGF-II cDNA contained a complete ORF consisting of 215 amino acids and one stop codon. Its molecular characteristics appear to be similar to the previously identified IGF-II molecules, in which a common primary structure exhibiting B, C, A, D, and E domains is evidently observed. This cDNA clone seems to be cleaved at $Ala_{52}$ for the $NH_2$-end signal peptide and appears to produce a 98 amino acid-long E-peptide from the $Arg^{118}$. The functional B-D domain regions, therefore, include 65 amino acids and is able to encode a 7.4-kDa protein. The most prominent structural difference between IGF-I and IGF-II was that the D domain of IGF-II exhibits a two-codon-deleted pattern compared to the 8 amino acid-containing IGF-I. The insulin family signature in the A domain and six cysteins forming three disulfide bridges between the B and A domains were evolutionary-conserved from teleosts to mammalian IGF-II. Interestingly, the E-peptide region appears to provide a distinct hallmark between teleosts in amino acid composition. The flIGF-II shows 85.1% of sequence identity to salmon and trout, 90.6% to tilapia, and 98.4% to perch in amino acid level. In tissue expressions of IGF-II, it is very likely that flIGF-II has a significant expression in the adult brain. However, liver seems to be the main source for IGF-II production, and relatively low signals were observed in the adult muscle and kidney. Taken together, it would be concluded that the functional region for IGF-II mRNA is highly similar in phylogeny and is evolutionary, conserved as a mediator for the growth of vertebrates.

• Korean Journal of Microbiology
• /
• v.30 no.1
• /
• pp.60-64
• /
• 1992

The secretion vector with promoter and signal sequence region of neutral protease gene (npr) from Bacillus amyloliquefaciens was constructed by the technique of polymerase chain reaction (PCR). A unique restriction iste was introduced into the 3' of the signal coding region by the synthesis of PCR primer. To demonstrate the function of cloned promoter and signal sequence, we used the E. coli .betha.-lactamase structural gene as a foreign gene. The signal sequence of .betha.-lactamase gene was deleted by Bal31 exonuclease and only mature region was introduced into the secretion vector. Bacillus subtilis cells transformed by the recombinant vector synthesized the fusion protein and were also capable of removing the signal peptide from the original fusion protein, as judged by the assay of .betha.-lactamase activity and secretion into the growth medium by western blotting.

• Journal of Microbiology and Biotechnology
• /
• v.32 no.3
• /
• pp.278-286
• /
• 2022

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.

• IMMUNE NETWORK
• /
• v.2 no.2
• /
• pp.102-108
• /
• 2002

Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2001.06a
• /
• pp.40-45
• /
• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.2
• /
• pp.600-608
• /
• 2013

Amide analogs of tridecapeptide ${\alpha}$-factor (WHWLQLKPGQPMYCONH$_2$) of Saccharomyces cerevisiae, in which Trp at position 1 and 3 were replaced with other residues, were synthesized to ascertain whether cooperative interactions between two Trp residues occurred upon binding with its receptor. Analogs containing Ala or Aib at position 3 of the peptide $[Ala_3]{\alpha}$-factor amide (2) and $[Aib_3]{\alpha}$-factor amide (5) exhibited greater decreases in bioactivity than analogs with same residue at position one $[Ala^1]{\alpha}$-factor amide (1) and $[Aib^1]{\alpha}$-factor amide (4), reflecting that $Trp^3$ may plays more important role than $Trp^1$ for agonist activity. Analogs containing Ala or Aib in both position one and three 3, 6 exhibited complete loss of bioactivity, emphasizing both the essential role and the combined role of two indole rings for triggering cell signaling. In contrast, double substituted analog with D-Trp in both positions 9 exhibited greater activity than single substituted analog with D-Trp 8 or deleted analog 7, reflecting the combined contribution of two tryptophane residues of ${\alpha}$-factor ligand to activation of Ste2p through interaction with residue $Tyr^{266}$ and importance of the proper parallel orientation of two indole rings for efficient triggering of signal G protein coupled activation. Among ten amide analogs, $[Ala^{1,3}]{\alpha}$-factor amide (3), $[Aib^{1,3}]{\alpha}$-factor amide (6), [D-$Trp^3]{\alpha}$-factor amide (8) and [des-$Trp^1,Phe^3]{\alpha}$-factor amide (10) were found to have antagonistic activity. Analogs 3 and 6 showed greater antagonistic activity than analogs 8 and 10.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.12
• /
• pp.2036-2045
• /
• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.