• BMB Reports
• /
• v.29 no.2
• /
• pp.137-141
• /
• 1996

A partial cDNA encoding a Korean radish isoperoxidase was obtained from a cDNA library prepared from 9 day old radish root. In order to obtain Korean radish isoperoxidase cDNA, 5' RACE (rapid amplification cDNA end) PCR was performed and a cDNA (prxK1) encoding a complete structural protein was obtained by RT (reverse transcription)-PCR. Sequence analysis revealed that the length of the cDNA was 945 base pairs, and that of the mRNA transcript was ca. 1.6 kb. The deduced amino acid of the protein were composed of 315 amino acid residues and the protein was 92% homologous to turnip peroxidase, and 46% to 50% homologous to other known peroxidases. The 945 bp cDNA encoding Korean radish isoperoxidase was overexpressed in Escherichia coli up to approximately 9% of total cellular protein. The recombinant fusion protein exhibited 43 kDa on SDS-PAGE analysis and the activity level of the recombinant nonglycosylated protein was two fold higher in IPTG induced cell extracts than that of uninduced ones.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.2
• /
• pp.375-382
• /
• 2010

The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei, contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers, and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein secretion in L. casei.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
• /
• 2014.05a
• /
• pp.813-815
• /
• 2014

Novel baculovirus vector systems including genes of polyhedron promoter, vesicular stomatitis virus G (VSVG), polyA, cytomegalovirus (CMV) promoter, enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD) were constructed. These recombinant baculovirus vector systems were transfected into diverse cells of 293T, HepG2, HFF, and Hur7 cells and compared the effects of gene transfer and expression of these vector systems with control vector. From the result, we confirmed that these recombinant baculovirus vector systems were more excellent than control vector in efficacy of gene transfer and expression.

• Tuberculosis and Respiratory Diseases
• /
• v.61 no.3
• /
• pp.239-247
• /
• 2006

Background: Culture filtrate proteins secreted by mycobacteria are thought to play an important role in inducing protective immunity and to develop new methods for diagnosing tuberculosis. Methods: A culture filtrate protein of M. avium that was strongly reactive with goat antiserum against M. intracellulare was constructed. Its homologous protein (TB-14) in M. tuberculosis was cloned, expressed and purified. The inductions of IFN-${\gamma}$ stimulated with $10{\mu}g$ of TB-14 recombinant protein and $10{\mu}g$ PPD were estimated by using whole bloods from seven PPD (-) subjects, seven PPD (+) healthy volunteers and nine tuberculosis patients. Results: M. avium culture filtrate protein was confirmed as a hypothetical protein that was termed contig 116. A novel 14-kDa recombinant protein (TB-14) of M. tuberculosis was composed of 148 amino acids, including 30 amino acids of the signal peptide, and it showed 78% homology with M. avium. In the PPD (+) healthy volunteers, recombinant TB-14 protein strongly induced the secretion of IFN-${\gamma}$ in whole blood cultures. Conclusion: These results suggest that TB-14 recombinant protein might play an important role in inducing cell-mediated immunity against tuberculosis. Furthermore, TB-14 protein antigen and its antiserum will be available for the development of new diagnostic tools for tuberculosis.

• Tuberculosis and Respiratory Diseases
• /
• v.58 no.2
• /
• pp.142-152
• /
• 2005

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

• Reproductive and Developmental Biology
• /
• v.36 no.3
• /
• pp.193-198
• /
• 2012

This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (~35 kDa), dimer (~70 kDa), trimer (~105 kDa) and hexamer (~210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (~300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher's study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (${\beta}$-MeOH), anionic detergent (SDS) and heat ($95^{\circ}C$) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.8
• /
• pp.1236-1241
• /
• 2007

Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form ${\alpha}$-helix structures are more translocated to the periplasm.

• BMB Reports
• /
• v.31 no.6
• /
• pp.585-589
• /
• 1998

The herpes simplex virus type-1 (HSV-1)-encoded DNA polymerase consists of two subunits, the products of the UL30 and UL42 genes. UL30 and UL42 were coexpressed in Sf9 cells infected with recombinant baculoviruses carrying the two genes. The UL30 and UL42 gene products remained tightly associated throughout the purification, which led to a near homogeneous heterodimer composed of the DNA polymerase and UL42 protein. The DNA polymerase-UL42 protein heterodimer, purified from the recombinant baculovirus-infected Sf9 cells, showed the same high degree of processivity of deoxynucleotide polymerization as the enzyme purified from the HSV-1 infected primate cells. Like the latter, it contained a 3'-5' exonuclease activity that specifically hydrolyzes an incorrectly matched nucleotide at the 3' terminus of a primer, thereby contributing to the fidelity of DNA replication.

• Journal of the Korea Institute of Military Science and Technology
• /
• v.14 no.4
• /
• pp.710-718
• /
• 2011

Edema factor(EF) is a portion of anthrax toxin which produces edema when combined with protective antigen. This paper describes about technique for cloning, expression, purification and activity test of EF. Using the E. coli expression system, we could make recombinant EF protein although it's origin is Bacillus anthracis. And also we could culture massively and purify highly pure protein. Finally we confirm a enzyme activity of purified EF to increase intracellular cAMP level. Through establishing this technique, it can be possible to research about EF in depth and apply to expression and purification of many other protein in biology.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.2
• /
• pp.308-311
• /
• 2006

In an attempt to increase production of LK8, an 86-amino-acid kringle fragment of human apolipoprotein(a) with three disulfide linkages, protein disulfide isomerase (PDI) was coexpressed in recombinant Saccharomyces cerevisiae harboring the LK8 gene in the chromosome. Whereas overexpression of the LK8 gene without coexpressing PDI was detrimental to both host cell growth and LK8 production, coexpression of PDI increased the LK8 production level by 2.5-fold in batch cultivation and 5.0-fold in fed-batch cultivation compared with the control strain carrying only the genomic PDI gene.