• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.993-1001
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• 2007

The technique of serological analysis of antigens by recombinant cDNA expression library (SEREX) uses autologous patient sera as a screening probe to isolate tumor-associated antigens for various tumor types. Isolation of tumor-associated antigens that are specifically reactive with patient sera, but not with normal sera, is important to avoid false-positive and autoimmunogenic antigens for the cancer immunotherapy. Here, we describe a selection methodology to isolate patient sera-specific antigens from a yeast surface-expressed cDNA library constructed from 15 patient lung tissues with non-small cell lung cancer (NSCLC). Several rounds of positive selection using patient sera alone as a screening probe isolated clones exhibiting comparable reactivity with both patient and normal sera. However, the combination of negative selection with allogeneic normal sera to remove antigens reactive with normal sera and subsequent positive selection with patient sera efficiently enriched patient sera-specific antigens. Using the selection methodology described here, we isolated 3 known and 5 unknown proteins, which have not been isolated previously, but and potentially associated with NSCLC.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.244-250
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• 2000

An ${\alpha}-glucosidase$ gene (aglA) from thermophilic Bacillus sp. DG0303 was cloned, sequenced, and expressed in Escherichia coli. The aglA was localized to the 2.1-kb PvuI-XmnI region within the 5.9-kb DNA insert of the gybrid plasmid pAG1. The gene consisted of an open reading frame of 1,686 bp with an unusual GTG initiation codon and TGA termination codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 562 amino acid residues with a M, of 66,551 dalton. A comparative amino acid sequence analysis revealed that DG0303 ${\alpha}-glucosidase$ is related to bacillary oligo-1, 6-glucosidases. The Bacillus sp. DG0303 ${\alpha}-glucosidase$ showed a high sequence identity (36-59%) to the B. flavocaldarius, B. cereus, and B. thermoglucosidasius oligo-1, 6-glucosidases. The number of prolines in theses four ${\alpha}-glucosidases. was observed to increase with increasing thermostability of these enzymes. The cloned ${\alpha}-glucosidase was purified from E. coli $DH5{\alpha}$ bearing pAG1 and characterized. The recombinant enzyme was identical with the native enzyme in its optimum pH and in its molecular mass, estimated by sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The temperature optimum of the cloned ${\alpha}-glucosidase$ was lower than that of the native enzyme.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.574-579
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• 1989

Chromosomal DNA fragments of Bacillus sp. YA-14, isolated from soil as a potent $\beta$-xylosidase producing bacterium, were ligated to a vector plasmid pBR322 and used to transfer Escherichia coli HB101 cells. The recombinant plasmid pYXL22 was found to enable the transformants to produce $\beta$-xylosidase. pYXL22 was found to contain the 7.0 kb HindIII DNA fragment originated from the Bacillus sp. YA-14 chromosomal DNA by Southern hybridization. The optimum temperature for the reaction of $\beta$-xylosidase produced by E. coli HB101 (pYXL22) was appeared at 3$0^{\circ}C$. The enzyme was maintained stably up to 4$0^{\circ}C$ when stored 1hr at 4$0^{\circ}C$. The $\beta$-xylosidase was repressed completely by 0.4% (w/v) glucose concentration in E. coli HB101 (pYXL22). The optimum concentration of xylose for the $\beta$-xylosidase production in Bacillus sp. YA-14 was 0.2% (w/v).

• Journal of Microbiology and Biotechnology
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• v.29 no.11
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• pp.1717-1728
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• 2019

The gene encoding β-galactosidase was cloned from Bifidobacterium longum RD47 with combinations of several bifidobacterial promoters, and expressed in B. bifidum BGN4. Among the recombinant bifidobacteria, BGN4+G1 showed the highest β-galactosidase level, for which the hydrolytic activity was continuously 2.5 to 4.2 times higher than that of BGN4 and 4.3 to 9.6 times higher than that of RD47. The β-galactosidase activity of BGN4+G1 was exceedingly superior to that of any of the other 35 lactic acid bacteria. When commercial whole milk and BGN4+G1 were reacted, BGN4+G1 removed nearly 50% of the lactose in the milk by the 63-h time point, and a final 61% at 93 h. These figures are about twice the lactose removal rate of conventional fermented milk. As for the reaction of commercial whole milk and crude enzyme extract from BGN4+G1, the β-galactosidase of BGN4+G1 eliminated 51% of the lactose in milk in 2 h. As shown below, we also compared the strengths and characteristics of the strong bifidobacterial promoters reported by previous studies.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.28-33
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• 1994

In normal cells tau protein is associated with axonal microtubules, whereas in Alxheimer's disease it is immobilized in the somatodendritic compartment of certain nerve cells as a major component of the paired helical filament. As a part of the study to analyze the nature of the paired helical filament (PHF) deposits and some related factors in brain, we have cloned and expressed a human tau gene cDNA in Escherichia coli to obtain the recombinant human tau protein in abundance.

• KSBB Journal
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• v.10 no.3
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• pp.264-270
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• 1995

Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.191-193
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• 2000

A whole-cell biocatalyst was constructed by immobilizing an enzyme on the surface of the yeast Saccharomyces cerevisiae. The gene encoding Bacillus macerans cyclodextrin glucanotransferase(CGTase) was fused with the AGA2 gene encoding a small peptide disulfide-linked to the aga1, a cell wall protein of a-agglutinin. The plasmid was introduced S. cerevisiae and expressed in the medium consisting of 10g/L yeast extract, 20g/L peptone, and 20g/L galactose. The activity was detected with the formation of cyclodextrin(CD) from 10g/L soluble starch. Surface display of CGTase was also verified with the halo-test, flow cytometry, and immunofluorescence microscopy. The recombinant S. cerevisiae produced ${\alpha}-cyclodextrin$ more efficiently than the free CGTase by simultaneous fermentation and cyclization as yeast consumes glucose and maltose which are inhibitors for CD synthesis.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• BMB Reports
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• v.39 no.5
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• pp.571-577
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• 2006

Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus ($N{\omega}V$). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of $Mg^{2+}$ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

• Parasites, Hosts and Diseases
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• v.43 no.3 s.135
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• pp.101-110
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• 2005

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited $42-57\%$ homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GOSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cock-roach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.