• Journal of Microbiology and Biotechnology
• /
• v.20 no.1
• /
• pp.54-62
• /
• 2010

A novel harpin-like protein, HpaXm, was described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum. The hpaXm was found to be localized between hrp2 and hrcC. A phylogenetic analysis of the complete amino acid sequence or solely the 13 highly conserved residues $H_2N$-SEKQLDQLLTQLI-COOH in the N-terminal $\alpha$-helix indicates that HpaXm is evolutionarily closer to HpaGXag and HpaXac than to Hpa1Xoo and Hpa1Xoc. A synthesized peptide containing two heptads, 39-LDQLLTQLIMALLQ-52, from the N-terminal a-helical region of HpaXm displayed comparable activity in inducing a hypersensitive response, but two other synthesized derivatives, $HpaXm{\Delta}T44C$ and $HpaXm{\Delta}M48Q$, showed reduced HR-triggering activity. The data from a GST trap test revealed that HpaXm was released into the extracellular medium, hpaXm mutant deficient for the leader peptide (1-MNSLNTQIGANSSFL-15) was unable to be secreted outside cells but still induced HR in tobacco leaves.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.5
• /
• pp.660-665
• /
• 2003

High-level expression of Thermus caldophilus GK24 alkaline phosphatase (Tca APase) was achieved in Escherichia coli using the pET-based expression plasmids, pEAP1 and pEAP2. In the case of plasmid pEAP2, the signal peptide region of Tca APase was replaced by the PelB leader peptide of expression vector pET-22b(+). Furthermore, the expression level was somewhat higher than that of plasmid pEAPl. A rapid purification procedure of Tca APase overproduced in E. coli was developed which involved heating to denature E. coli proteins followed by HiTrap Heparin HP column chromatography. Optimal temperature and pH and $Mg^{2+}$ dependence of the recombinant Tca APase were similar to those of native enzyme isolated from T. caldophilus GK24.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 1995.06b
• /
• pp.97-117
• /
• 1995

Copper resistance in Pseudomonas syringae pv. tomato is determined by copper-resistance operon (cop) on a highly conserved 35 kilobase plasmid. Copper-resistant strains of Pseudomonas syringae containing the cop operon accumulate copper and develop blue clonies on copper-containing media. The protein products of the copper-resistance operon were characterized to provide an understanding of the copper-resistance mechanism and its relationship to copper accumulation. The Cop proteins CopA (72 kDa), CopB (39 kDa), and CopC (12 kDa) were produced only under copper induction. CopA and CopC were periplasmic proteins and CopB was an outer membrane protein. Leader peptide sequences of CopA, CopB, and CopC were confirmed by amino-terminal peptide sequencing. CopA, CopB, and CopC were purified from strain PT23.2, and their copper contents were determined. One molecule of CopA bound 10.9${\pm}$1.2 atoms of copper and one molecule of CopC bound 0.6${\pm}$0.1 atom of copper. P. syringae cells containing copCD or copBCD cloned behind the lac promoter were hypersensitive to copper. The CopD (32 kDa), a probable inner membrane protein, function in copper uptake with CopC. The Cop proteins apparently mediate sequestration of copper outside of the cytoplasm as a copper-resistance mechanism.

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.1
• /
• pp.146-152
• /
• 2004

Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors containing the cDNA of swine mature ghrelin (pGEM-wt-sGhln, pGEM-wt-hGhln), ghrelin mutant of $Ser^3$ with $Trp^3$ (pGEM-mt-sGhln, pGEM-mt-hGhln) and truncated ghrelin derivative (pGEM-tmtsGhln, pGEM-tmt-hGhln) encompassing the first 7 residues of ghrelin (including $Ser^3$ substituted with $Trp^3$) and adding a basic amino acid, Lys (K) in the C-terminus. The constructs, pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-tmt-sGhln were linked with the ghrelin leader sequence, while the pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln were linked with a leader sequence from the human growth hormone releasing hormone (hGHRH). Intramuscular injection of 200 ${\mu}g$ pGEM-wt-sGhln or pGEM-tmt-sGhln augmented growth over 3 weeks in normal rats and peaked at day 21 or 14 post-injection respectively, whose body weight gains were on average approximately 6% or 19% heavier over controls. However, other injectable vectors had no such enhanced growth effects. Our results suggested that the efficacy of the ghrelin leader sequence was more effective than that of hGHRH in our system. Moreover, the results indicated that skeletal muscle might have the ability to posttranslationally modify the in vivo expressed ghrelin. And the most strikingly, the short ghrelin analog seems to mimic the biological effects more efficiently when compared with the full-length ghrelin.

• Journal of Life Science
• /
• v.9 no.4
• /
• pp.341-347
• /
• 1999

Insects use chitinolytic enzyme to digest chitin in the exoskelton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the silkworm, Bombyx mandarina, compared its sequenced with genes encoding chitinolytic enzymes from other sources. The insert DNA in the clone is 2,675 nucleotides long with an open reading frame of 1,695 uncletides that encodes a protein of 565 amino acids with a molecuar weight of 63.4 kDa. The 3' -untranslated region of 889 nucleotides is AT-rich and contains two putative polyadenylation signals. The N-terminal sequence of the encoded protein contains numerous hydrophobic residues characteristic of a leader peptide. The amino acid alignment revealed that the endo-$\beta$-N-acetylglucosaminidase had 83% and 97% homology with M. sexta and B. mori, respectively. The deduced amino acid had two highly conserved region at the amino acid residues 97-111 and 139-148 that were related to the existing chitinase.

• YAKHAK HOEJI
• /
• v.35 no.1
• /
• pp.22-29
• /
• 1991

pMB4 is a 2.4-kilobase plasmid of Staphylococcus aureus TR-1 that confers inducible resistance to the macrolide-lincosamide-streptogramin B(MLS) antibiotics. By subcloning studies, it was found that the MLS resistance determinant was located at 1.0Kb fragment between Sau3AI and TaqI sites. DNA sequence of the MLS resistant determinant, named ermC-4 was determined, and found to be highly homologous with that of ermC. Because the leader peptide sequence of ermC-4 was identical with that of ermC, the expression of the resistance gene is thought to be controlled by posttranscriptional attenuation in S. aureus TR-1.

• Korean Journal of Microbiology
• /
• v.46 no.1
• /
• pp.68-72
• /
• 2010

The ${\beta}$-1,4-glucanase gene of Bacillus licheniformis B1 was expressed in Esherichia coli BL21, and a protein with a mass of 50 kDa that was soluble was overproduced. A protein with a mass of 37 kDa was secreted from B. licheniformis. It seems that the ${\beta}$-1,4-glucanase produced in E. coli contained the leader peptide and unprocessed carboxy-terminal region, but its processing occurred in the carboxyterminal in Bacillus. The optimal temperature of ${\beta}$-1,4-glucanase was $40^{\circ}C$. The enzyme still had 76% maximal activity at $60^{\circ}C$. The optimal pH of the enzyme was 7. The enzyme retained considerable activities over the weak-acidic, neutral, and weak-basic pH range. Acidic fungal cellulases are used in food, detergent, pulp, paper, textile industries. However, studies about neutral and alkaline cellulase are not enough. The cellulase developed in this study may be useful for industrial applications in the fields of biofuel development.

• Molecules and Cells
• /
• v.26 no.2
• /
• pp.140-145
• /
• 2008

Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides ($OmpASP_{tr}$) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP ($OmpASP_{tr}$) and a factor Xa cleavage site (Xa) [$OmpASP_{tr}{\mid}(xxx){\mid}Xa$], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ HepI fusion proteins revealed that the transmembrane-like domains derived from the $OmpASP_{tr}{\mid}(xxx){\mid}Xa$ sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.468-472
• /
• 2003

Recently, genetic engineering techniques have been used to display various heterologous peptides and proteins (enzyme, antibody, antigen, receptor and fluorescence protein, etc.) on the yeast cell surface. Living cells displaying various enzymes on their surface could be used repeatedly as 'whole cell biocatalysts' like immobilized enzymes. We constructed a yeast based whole cell biocatalyst displaying T. reesei cellobiohydrolase I (CBH I ) on the cell surface and endowed the yeast-cells with the ability to degrade cellulose. By using a cell surface engineering system based on ${\alpha}-agglutinin,$ CBH I was displayed on the cell surface as a fusion protein containing the N-terminal leader peptide encoding a Gly-Ser linker and the $Xpress^{TM}$ epitope. Localization of the fusion protein on the cell surface was confirmed by confocal microscopy. In this study, we report on the genetic immobilization of T. reesei CBH I on the S. cerevisiae and hydrolytic activity of cell surface displayed CBH I.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.7 no.1
• /
• pp.38-42
• /
• 2002

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from the Serratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids. Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide and N-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a $98\%$ similarity to that of S. marcescens OMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N'-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount of N-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer $(analogue\;of\;NAG_2)$, thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were $50^{\circ}C$ and 5.0, respectively.