Earticle

Codonopis lanceolata, a perennial herb, belongs to Campanulaceae. Codonopis lanceolata radix has been utilized as a seasonal food and traditional tonic medicine for the past years in Korea. Codonopis lanceolata which contains is known to be effective for the discharge of phlegm, counteracting poison, asthma, phthisis tuberculosis, etc. This study was concentrated to investigate the identification of anti-allergic effect of various extracts from Codonopis lanceolata. We used the cascade extraction procedure with various solvents from hot-water extract to obtain several solvents fractions and crude polysaccharides from Codonopis lanceolata. Mast cell releases various mediators upon stimulation. Stimulation of mast cell with compound 48/80 initiates the activation of a signal transduction pathway, leading to histamine release. In pathological skin condition, histamine is involved in the induction of itching and edema. Therefore, to identify the anti-allergiceffect, cytotoxicity was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Histamine content was measured using o-phthalaldehyde by spectrofluorometric procedure.

Pseudomonas putida KT2440 zwf, encoding G6PDH catalyzes the oxidation of glucose-6-phosphate to 6 phosphoglucolactone. Cultured in minimal medium containing glucose, northern blot analysis has shown that the zwf-1 was highly induced. However the expression level was not detected in the presence of pyruvate or succinate. We constructed a GFP-based reporter fusion using the zwf-1 gene to study the activity of the promoter. Pzwf-1 is increased in the presence of glucose. In contrast with glucose, organic acids suppress the promoter activity. A knock out mutant of HexR, a putative transcription regulator adjacent to zwf-1 gene, has constant Pzwf-1 activity in any condition. Electrophoretic mobility shift assay shows that the HexR protein binds the zwf-1 promoter region. Interestingly, the zwf-1 is also highly expressed in the presence of ROS producing materials. The Zwf induction mechanism caused by oxidative stress is not known in Pseudomonas sp. Therefore induction of zwf by oxidative stress of P. putida will be discussed. This work was supported by a NCRC grant (R15-2003-012-02002-0) and a grant (R0503443) from the BioGreen21 program

E. coli is one of the most preferred strains for the production of recombinant proteins because of many reasons, such as its fast growth rate and many available genetic tools. Still, there are several problems in using E. coli strain as a microbial protein factory. Formation of inclusion body is one of problems to overcome, which is frequently observed when the recombinant proteins are highly expressed. We hypothesized that the knockout of a specific gene could reduce the formation of inclusion body through direct or indirect impacts on protein synthesis rate in E. coli. As such, we investigated the relationships between a specific genotype and inclusion body forming phenotypes. Specifically, we transformed a plasmid containing a reporter protein (GFP) under the control of a strong inducible promoter into a systematic knockout library. The transformed knockout library were screened by FACS and microplate reader. Based on the intensity of the fluorescence of GFP, we selected knockout mutants which accumulated higher amount of soluble GFP as compared to the parent strain. Accumulation of higher amounts of soluble GFP in knockout mutant was confirmed using SDS-PAGE. These results suggest that the single knockout of the specific gene can affect the formation of inclusion body in E. coli.

Lambda phage-originated recombination functions, collectively called Red, enable simple and specific in vivo gene manipulation in E. coli, which is of great use in functional genomics, BAC engineering and gene therapy. Directly from PCRs, linear dsDNAs flanked by short homology arms can be used as recombination substrates with high efficiency. However, their fundamental mechanisms remain to be scrutinized unambiguously for correct design of substrates and delicately-ramified applications. We examined the influence of terminally-linked non-homology on recombination functions, from which a strategy to show the validity of a ‘replication-dependent annealing’ model proposed previously was built: a linear dsDNA molecule harboring three homology region and two antibiotic resistance genes. Notably, its 3’ single stranded overhang complementary to lagging strand template of bacterial chromosome was most favorably annealed to the targets. Probably, it is caused by sequential exposure of chromosomal homology regions as the replication fork advances progressively, with lagging strand homology being unwound first. This bias was extensively observed throughout the chromosome, supporting the availability of the proposed model.

The purpose of this study was to investigate the effect of low molecular peptides of Asterias amurensis on skin immune activation. This peptides were isolated form A. amurensis by enzymetic method with ultrasonication. The inhibitory effect of the peptides on hyaluronidase and PGE2 in UV-irradiated human dermal fibroblast were also performed. Among the peptides, F4(38 kDa) showed the strongest PGE2 and Hyaluronidase inhibitory activities1). The F4(38 kDa) peptides reduced nitric oxide production about 250% by adding 1.0 ㎎/㎖ compared with LPS(lipopolysaccharides). The cytotoxicity of peptides at 1.0 ㎎ /㎖ was below 15%. At 0.2 ㎎/㎖ of the F4(38 kDa) peptides, PGE2 expression2) was inhibited significantly compared with control. These results imply that the F4(38 kDa) peptides could be used as a new immuno-modulating agent for the skin.

The ketolides, the recent generation of antimicrobials derived semi-synthetically from the 14-membered ring macrolide erythromycin A, are characterized by possessing a 3-keto group in place of an L-cladinose group of erythromycin A. The semi-synthetic steps of ketolides involving protection and deprotection of functional groups can be shortened by combinatorial biosynthesis; namely, the employment of 5-O-desosaminyl erythronolide A (DesEA) as a precursor in the ketolide synthesis reduces the steps for the removal of L-cladinose attached at the C-3 position in erythromycin A. Deletion of an eryBV gene in an erythromycin-producer Saccharopolyspora erythraea that encodes glycosyltransferase acting on erythronolide B by attachment of L-mycarose, resulted in the accumulation of 5-O-desosaminyl erythronolide B (DesEB). In vivo expression of a gene pikC from the pikromycin biosynthetic pathway of Streptomyces venezuelae that encodes a substrate-flexible cytochrome P450 monooxygenase in the eryBV deletion mutant strain of Sac. erythraea successfully generated DesEA.

The Pseudomonas strain conserves a defense mechanism for the oxidative stress including the superoxide dismutase and catalase systems1-4). The genes for the defense mechanism, sodB and katA from an organic solvent tolerant Pseudomonas isolate were synthesized by PCR and cloned into pGEM-T easy vector for phylogenetic analysis. As a result, the sodB sequence showed more than 80% identity, in comparison with that of the Aeromonas species including A. bestiarum, A. hydrophila, A. bestiarum, A. media, and A. bestiarum, respectively. The nucleotide sequence of the sodB was also significantly homologous to that of P. putida, P. entomophila, P. fluorescens, P. aeruginosa, P. mendocina, and P. syringae, respectively. The katA sequence from the isolate was, of note, highly homologous to the catalase gene sequence of Aeromonas hydrophila subsp. Hydrophila, scoring 90% identity. This finding may give a clue to the significant phylogenetic relationship among the sodB sequence of Pseudomonas putida and Aeromonas species. High level expression and purification of the protein, superoxide dismutase B using the expression vector system is under the study.

Sugar polymer has been considered as a biomaterial for medical applications. These biomaterials are widely used in the preparations of burn dressing, artificial membranes and contact lens. In particular, characteristics of contact lens material are moisture content, oxygen permeability, oxygen transmissibility, equivalent oxygen percentage, surface wet ability and refractive index. Hydrogels defined as materials that exhibit the ability to swell in water and retain a significant fraction of water within their structure. Hydrogel may be chemically stable, degradable, eventually disintegradable, and dissolvable. In this study, the objective of experiment is to verify contact-lens properties of poly(sorbitan methacrylate) hydrogel synthesized. Poly(sorbitan methacrylate) is manufactured by free radical polymerization from sorbitan methacrylate as monomer.

In the present study, we performed a differential proteomic analysisusing two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry to clarify the molecular mechanism for suppressive effect of chitosan oligosaccharide (CO) during differentiation of adipocyte 3T3-L1.1),2) The cell differentiation was significantly inhibited by CO at the concentration of 4 mg/mL. The protein mapping of adipocyte homogenates by 2-DE revealed that numerous protein spots were differentially altered in response to CO treatment. Out of 50 identified proteins showing significant alterations, 6 were up-regulated and 44 were down-regulated by CO treatment in comparison to control mature adipocytes. Among them, most of the proteins are associated with lipid metabolism, cytoskeleton, and redox regulation. Taken together with immunoblot analysis, it was concluded that the inhibitory effect of CO on adipocyte differentiation was mediated by C/EBPα and PPARγ pathway through significant down-regulations of important adipogenic molecules such as fatty acid binding protein and glucose transporter 4.

Ferredoxin-NADP+ reductase (Fpr) is known to control NADP+/NADPH pool in bacteria. Interestingly, P. putida have two Fpr (FprA, FprB) and its two cognate [2Fe-2S] Ferredoxins (FdA, FdB). Kinetic study of each Fpr was performed using a variety of assays [diaphorase, cytochromeC and ferric reductase assay]. Yeast two-hybrid was used to determine in vivo interaction between Fpr and Fd. Likewise Fpr of other bacteria, both FprA and FprB can use DPIP and ferricyanide as the terminal electron acceptor, which indicated that both Fprs have diaphorase activity. We confirmed that both Fpr products prefer NADPH to NADH at the redox complex formation. In the cytochromeC and ferric reductase assay, complex between FprB and FdA has high catalytic activity (kcat/Km) which is 5-fold higher than that of other redox couples. Similarly, In vivo interaction study using yeast two hybrid system confirms that FprB has more specific binding activity with FdA than with FdB. The data shows that specific interaction between Fpr and its cognate partner is required for efficient catalytic function. This work was supported by a NCRC grant (R15-2003-012-02002-0) and a grant (R0503443) from the BioGreen21 program

Recent studies have raised industrial application of Mannheimia succiniciproducens to produce succinic acid.1) Shuttle vectors replicating in E. coli and M. succiniciproducens were constructed. These vectors were present at ca. 10 copies per cell and stably maintained in M. succiniciproducens during the serial subculturing without antibiotic for 48 generations. By optimizing the electroporation conditions, the transformation efficiencies of 3.0×106 transformants/mg DNA were obtained in M.succiniciproducens. In addition, there were compatible restriction and modification systems judged from the transformation efficiencies in E. coli K strain and M. succiniciproducens. DNA fragments up to 8.2 kb in size can be cloned into shuttle vectors. Thus, the shuttle vectors developed in this study are useful tools for genetic and metabolic engineering of rumen bacteria. (This work was supported by the Genome-based Integrated Bioprocess Development Project (No. 2005-01294) of the Ministry of Science and Technology through the Korea Science and Engineering Foundation (KOSEF). Further supports by the LG Chem Chair Professorship, IBM SUR program, and by the KOSEF through the Center for Ultramicrochemical Process Systems are appreciated.)

Outbreak of antibiotics resistant bacteria is a serious concern to medical, pharmaceutical, and food industry. While there are many accumulating evidences and speculations on how bacteria develop resistance to antibiotics, systems-level cataloging of potential resistance mechanisms has not been done. In this study, we attempted to identify putative gene targets that induce antibiotics resistance using a systematic knockout collection. Specifically, an E. coli knockout library based on the Keio collection was challenged with various concentrations of ampicillin in order to isolate ampicillin resistant knockout mutants. Of 48 colonies grown on various ampicillin concentrations, seven knockout mutants exhibiting significant resistance were isolated. To confirm that the resistance phenotype was caused not by spontaneous mutations but by a known single gene mutation, the mutations (single gene knockout) from the seven mutants were transferred into E. coli DH5α strain having a different genotypic background by P1 transduction. All of the identified single gene knockouts conferred ampicillin resistance into the E. coli DH5α

Redesign of the existing natural biological systems requires many components that can be controlled by purpose. Protein functions as well as regulation mechanisms, first of all, should be understood in a systematic manner for well-controlled system. Although many methods are already used to clarify protein functions, they have a limitation on the high-throughput analysis by reason of the labor-intensive and time-consuming process. In this study, we developed a rapid and simple method to analyze protein functions efficiently based on the PCR-based site-directed mutagenesis and the expressional PCR using a coupled in vitro transcription/translation system derived from E. coli and eGFP (enhanced green fluorescence protein) gene as a template.1-2) Various deletion mutants showed different fluorescence activity. The results also showed that this method allows a rapid and simple route for the functional genetics.

Mannheimia succiniciproducens MBEL55E, a capnophilic rumen bacterium, produces succinic acid as a major fermentation product during anaerobic cultivation. M. succiniciproducens has been known to use anaplerotic pathway to produce succinic acid. In the starting point of the anaplerotic pathway, two Enzymes, phosphoenolpyruvate carboxylase(Ppc) and phosphoenolpyruvate kinase(PckA), convert phosphoenolpyruvate to oxaloacetate. We analysed activation of both enzymes to elucidate the roles in the production of succinic acid. This study shows that PckA is 50 times stronger than Ppc in M. succiniciproducens, suggesting that PckA is a very important enzyme for producing succinic acid. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (2005-01294). Further supports by the LG Chem Chair Professorship, IBM SUR program, Microsoft, and by the KOSEF through the Center for Ultramicrochemical Process Systems are appreciated.].

New strain development strategy using kinetic models and metabolic control analysis was investigated. In this study, previously reported mathematical models describing the enzyme kinetics of intracellular threonine synthesis were modified for mutant threonine producer Escherichia coli TF5015. Using the modified models, metabolic control analysis was carried out to identify the rate-limiting step by evaluating the flux control coefficient on the overall threonine synthesis flux exerted by individual enzymatic reactions. The result suggested the production of threonine could be enhanced most efficiently by increasing aspartate semialdehyde dehydrogenase (asd) activity of this strain. Amplification of asd gene in recombinant strain TF5015(pCL-ParoF-asd) increased the threonine production up to 23%, which is much higher than 14% obtained by amplifying aspartate kinse (thrA), other gene in threonine biosynthesis pathway.

L-Arabinose is a major composition of some plant materials; therefore the metabolic pathway of L-arabinose has been identified mainly in microbes which degrade plant materials. Currently, three different pathways for L-arabinose degradation are known. Many bacteria including Escherichia coli convert L-arabinose to D-xylulose phosphate, which is entered into the pentose phosphate pathway. Some fungi convert L-arabinose to L-arabinitol by using aldose reductase and then D-xylulose phosphate is produced. In addition, a recent study showed that Azospirillum brasiliense converts L-arabinose to α-ketoglutarate via non-phosphorylated pathway composed of five enzymatic steps. The first step of this pathway is catalyzed by L-arabinose dehydrogenase (Abra_AraDH).1) Recently, we found a novel L-arabinose dehydrogenase (Bcep_AraDH) from Burkholderia cepacia KCTC 2966. To characterize this enzyme, the gene encoding B. cepacia L-arabinose dehydrogenase was cloned and expressed in E.coli BL21(DE3). The enzyme was purified using Ni-NTA column and its kinetic parameters were determined using various sugars as substrates. There was no sequence homology between Abra_AraDH and Bcep_AraDH. It was also found that Bcep_AraDH could utilize five different sugars; L-arabinose, D-galactose, D-fucose, D-glucose and D-xylose. We postulated that the enzyme specificity is related to the structural property of sugars

1,2-propandiol(1,2-PD) is used in unsaturated polyester resins, cosmetics, pharmaceuticals and as an environmentally friendly antifreeze and deicer1. In this study Saccharomyces cerevisiae YPH500, E. coli MG1655 (containing mgs gene), and Citrobacter freundii (containing dhdD gene) were used as the construction sources of genes and plasmids. In the previous study we could develop new Saccharomyces cerrevisiae strains which can produce 1,2-PD. 1,2-PD productivity in Saccharomyces cerevisiae strain seemed to be increased by constructing co-expression vectors2;; The pESC-URA vector containing two promoters, GAL1 and GAL10, which differs in the direction of transcription was used in the co-expression. Furthermore, the increase of 1,2-PD yield was induced, by adding input time and the amount of galactose which is controlled by the GAL promoter, to the fermentation process.

Systematic analysis using genome-scale metabolic network model has currently being developed to improve bioproduct yields and combine various high-throughput experiment data. Strategies for identifying gene deletions on genome-scale model were successfully applied and predict the target genes to achieve high amount of production. However, the systematic strategy for the gene amplification responsible to the increasing flux was not developed yet. Thus, using flux scanning with enforced objective flux (FSEOF) the genome scale model for valuable metabolite production was developed for the amplification of genes in the metabolic network. For validating the effectiveness of this approach, we carried out this approach was successfully employed to improve the yield of the production of shikimic acid, lycopene, and recombinant proteins. We anticipate that FSEOF plays an important role in the systematic strain improvement. [This work was supported by the Korean Systems Biology Research Program (M10309020000-03B5002-00000) of the Ministry of Science and Technologythrough the Korea Science and Engineering Foundation. Further supports by the BK21 program, LG Chem Chair Professorship, and IBM SUR program are appreciated.]

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell and its susceptibility to proteolytic degradation. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion peptide reached a maximum with a 12-mer of a histonin gene. Because of the RLKR residues present at the C-terminus of histonin, Furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 L of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.

Transmissible spongiform encephalopathies, including bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, are fatal neurodegenerative disease. The protein-only hypothesis holds that prion proteins are the infectious agents of transmissible spongiform encephalopathies. Two molecular forms of the prion protein, the infectious form (PrPSc) and the normal cellular form (PrPC), are believed to differ only by their conformation. PrPSc has been found to differ from PrPC by infectivity, an increased β-sheet content, an increased resistance to proteinase K and an oligomeric state rather that a monomeric state. In the prion protein, there is only one intramolecular disulfide bond that is believed to be changed to intermolecular linkage during the conversion of cellular form of prion protein (PrPC) into scrapie form (PrPSc). Thus, transient disulfide reduction is required, in which process the structural transition is the aim of this study. Fluorescence quenching, circular dichroism and electron paramagnetic resonance spectroscopy assay show that a-helical structure of PrPC becomes unstructured when disulfide bond is reduced. This state was aggregation-prone in water phase but becomes stabilized in the presence of membrane due to its membrane binding ability. Finally, the deposition of prion protein in the membrane destabilized membrane integrity.

S-Adenosylmethionine (SAM) is a cofactor for methyl group transfer in both prokaryotic and eukaryotic organisms and produced by S-Adenosylmethionine synthetase.1) To investigate the characterization of SAM-s from Pichia ciferrii for further applications, we cloned SAM-s from P. ciferrii by RACE, RNA capfishing and DNA walking. Cloned SAM-s encodesa 383 amino acids and 42KDa of size. After comparison of known other SAM-s sequences, cloned SAM-s showed about 90% of match with yeast SAM-s. The cloned SAM-s also contains consensus ATP-binding motif (G-X-G-X-X-G) and metal binding site (23D and 279D for Mg2+ and 51G for K+) which are located in other known SAM-s genes.2) Cloned SAM-s was inserted into E. coli expression vector to confirm their activity. After transformation, SDS-PAGE analysis was performed and we confirmed that cloned SAM-s was successfully expressed. However, no activity of cloned SAM- in E. coli was observed. Thus, cloned SAM-s was expressed in Pichia sp. for confirmation. The enzyme activity will be measured and result will be discussed.

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia. Diabetes mellitus ruins the kidney functions and leads to the development of chronic renal disease. Although the pathogenic mechanism of diabetic renal disease has not been elucidated, cytokine-driven proliferative and inflammatory mechanisms have been suggested to contribute to its progression.(1) Retinoid, a known anti-proliferation and anti-inflammation agent, has been reported to be beneficial in some experimental models of renal disease.(2) To find the effect of retinoid feeding in streptozotocin (STZ)-induced diabetic renal disease, we performed proteome analysis of rat kidney by the 2-dimensional-electrophoresis (2-DE). In this study, rats were divided into two groups; one group for STZ-injected rats + retinoid (retinol) supplement feeding and the other group for STZ-injected rats + general feeding. 4 weeks after STZ injection, kidney samples of all rats were homogenized and then analyzed by the 2-DE analysis. In conclusion, 8 proteins were up-regulated and 3 proteins were down regulated in retinoid supplement feeding diabetic rats compared to general feeding diabetic rats. Further studies of these proteins will help to understand comprehensive retinoid effects on kidney.

Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives aerobically at 59˚C and pH 2. The genome of T. acidophilum contains 1,564,905 bp on a single chromosome and encodes 1,509 ORFs, including a complete protein degradation pathway and various transport proteins1). Unfortunately, however, as much as 45% of its genome was annotated as hypothetical proteins. Hereby, we investigated how many intracellular proteins are expressed in D-glucose medium by means of 2D gel electrophoresis and gel-free shotgun proteomics. Among more than 300 spots visualized in 2D gels, 190 spots could be analyzed by MALDI-TOF MS/MS. Additionally, 381 different proteins were identified by gel-free LC-MS/MS. When the generated data were searched using MASCOT and Phenyx database, we were able to identify 470 different proteins in total. When identified proteins are divided into subgroups with regard to their functional categories, energy production and conversion is the largest group occupying 23% protein. Interestingly, 53% of the identified proteins have been detected at more than one position on the 2D electrophoresis gels, which suggests that these proteins are subject to regulation by PTMs.

Single chain Fv antibody (scFv) against c-Met is expected to be employed in clinical treatment or imaging of cancer cells due to the important biological roles of c-Met in the proliferation of malignancies. Hence it would be of importance to obtain a large amount of the recombinant anti-c-Met scFv for its practical utilization. Here, we show that the productivity of scFv against c-Met is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders, i.e. VL-linker-VH and VH-linker-VL, expressed them in the cytoplasm of E. coli trx/gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with VH/VL orientation was more than five times higher than that with VL/VH format. Coexpression of DsbC enhanced the yield of soluble amount of anti-c-Met scFv protein for both constructs. The scFv antibodies of the two different formats were purified through the interaction between Ni2+ and histidine tag on the c-terminus of recombinant protein, exhibiting almost same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with VH/VL or VL/VH formats and obtained similar results with the case of scFv antibodies.

L. mesenteroides B-1299CB4 utilizes sucrose for the synthesis of a dextran with only α -(1→6) glucosidic linkages. Comparison of the amino acid (aa) sequence of DSRBCB4 (a extransucrase of B-1299CB4) with glucansucrases from Streptococcus and Leuconostoc identified conserved aa residues in the catalytic core critical for enzyme activity. To mimic the reuteransucrase sequence, S642N/E643N/V644S was constructed. Triple mutant influenced against the product specificity converting the formation of an α-(1→6) dextran to that of a branched glucan [α-(1→3) and α-(1→4)]. Triple mutant increased levels of isomaltose and branched DP3 and DP4 oligosaccharides production compared to wild type. Triple mutant even showed higher leucrose yield up to 2-fold when incubated with sucrose alone. In the presence of maltose, triple mutant also formed panose as the most abundant accptor reaction product, 1.5-fold increased synthesis compared to wild type. Interestingly, triple mutant also synthesized unknown oligosaccharides from maltose acceptor reaction. When isomaltose was used as an acceptor, triple mutant showed 2.5-fold increased synthesis of isomaltotriose but without synthesis of isomaltopentaose

Recently, the genome sequence of hyperthermophilic archaeum Aeropyrum pernix K1 was identified and revealed an existence of a gene which had high sequence similarity (58% and 68%) to the gene encoding the thermosome A (ths A) and the thermosome B (thsB) in Pyrodictium occultum, respectively [1]. The two archaea chaperonin genes (ApcpnA, ApcpnB) were amplified by PCR and subcloned into the pET vector. When the constructed plasmids, pET3d-ApcpnA and pET21a-ApcpnB, were introduced into E. coli Rosetta (DE3) and BL21 CodonPlus (DE3), respectively. When the cell lysate of E. coli transformant cells were treated by heatshock at 90℃ for 20 min, the purification yield for ApCpn was higher than 80%. The supernatant was loaded on a HiTrap Q column equilibrated in buffer A (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, 20% glycerol) and the bounded ApCpn was eluted with a linear gradient of 0∼1.0 M NaCl. The transformant cells expressed ApCpn as a major intracellular soluble protein. A purified ApCpn showed an ATPase activity at 37 ∼ 85℃ and the thermal aggregation of citrate synthase, as a model protein, was significantly prevented in the presence of ApCpn and ATP.

Expression of recombinant proteins in Escherichia coli is often carried out at low temperature due to the improvement of solubility and stability of target protein at low temperature. However, the use of low temperature significantly limits the growth rate of expression host, leading to a reduction in the productivity of target protein. Here, we show that the introduction of 5' untranslated region of cspA mRNA (cspA 5'-UTR) into the transcript of a target gene enhances the expression level of target protein derived by T7 promoter at low temperature. First, the effects of cspA 5'-UTR sequence and CspA coexpression on the production rate of target protein were investigated by using green fluorescence protein (GFP) as a model. While the cspA 5'-UTR sequence had no influence on the expression rate of GFP at 37℃, it improved the expression level of GFP by approximately 1.5 fold at 15℃. Coexpression of CspA protein further slightly increased the expression level of GFP only when the cspA 5'-UTR was introduced into the gfp gene. Subsequently, the effect of the cspA 5'-UTR on the productivity of target protein was examined for six proteins (CysS, HisS, ProS, ThrS, TrpS, YadB) at 15℃, showing that the effect of cspA 5'-UTR was varied depending on target genes.

For the production of 1,2-propanediol, in Saccharomyces cerevisiae, pESC-URA including two multiple cloning sites was used as yeast expression vector. The vector is under the control of GAL promoters, GAL1 and GAL10 and the utility of the GAL promoters depends on the level of a positive activator [1],[2]. The metabolic pathway of 1,2-propanediol in yeast has been studied [3]. We selected two target gene, mgs and gldA, have important roles and cloned the target genes on pESC-URA vector. After transformation into S. cerevisiae by Li-AC method, the transformants on SDC URA- plate was selected. The concentrations of galactose and glucose were determined by expressing β-galactosidase gene on site 1 or 2 and both site 1 and 2 and assaying β -galactosidase activity [4]. As a results, S. cerevisiae containing plasmid pESC-URA grew well under the condition 2.5:17.5 (glucose: galactose).

Bacteria sense their environmental condition by two-component systems, and alter their metabolic behavior to adapt new environment. Two-component systems consist of histidine kinase (HK) and response regulator (RR) components. The HK autophosphorylates a conserved histidine residue in response to an environmental stimulus. The phosphate is transferred to a conserved aspartic acid residue in the RR, and then transcription of appropriate genes is activated. Behalf of complete genome sequence projects of living organisms, whole genome based evolutionary analysis, which can give more global and integrative insight than single gene based evolution analysis, became possible.1,2) In this study, two-component systems of 79 bacteria was collected and evolutionary relationship between two-component systems was evaluated. [This work was supported by the Korean Systems Biology Research Grant (M10503020001-07N0302-00112) of the Ministry of Science and Technology.]

The pYES2/CT and pYES3/CT, yeast expression vectors, are used for 1,2-propanediol production. Maximum β-galactosidase specific activity was assayed using the pYES2/CT/lacZ and pYES3/CT/lacZ for determination of harvest time. The pYES2/CT and pYES3/CT are 6.0 kb and 5.9 kb vector, respectively. The vectors contain the 2μ origin for maintenance and replication in yeast1). The pYES2/CT and pYES3/CT contain URA3 gene and TRP1 gene marker for selection, respectively. The vectors contain the GAL1 promoter. The GAL1 promoter require addition of galactose for induce expression of protein2). The effect of medium composition, glucose and galactose concentration on LacZ gene expression level were investigated. Assay of β-galactosidase specific activity study performed with Saccharomyces cerevisiae KCCM 50515. β -galactosidase assay was performed by ο-nitrophenyl-β-D-galactopyranoside (ONPG)3). Maximum β-galactosidase specific activity was obtained after 41 h of growth in YPDG medium containing 1.5% glucose and 0.5% galactose.