• Journal of Microbiology
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• 제45권6호
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• pp.528-533
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• 2007

In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementary-determining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.

• 대한미생물학회지
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• 제1권1호
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• pp.27-28
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• 1958

• BMB Reports
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• 제39권1호
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• IMMUNE NETWORK
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• 제2권2호
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• pp.91-95
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• 2002

Background: In order to characterize human antibodies with specificity for mite allergens at the molecular level, a scFv phage display library was constructed using peripheral blood mononuclear lymphocytes from an asthma patient allergic to mite as Ig gene sources. Methods: Immunoglobulin $V_H$ and V gene fragments were obtained by polymerase chain reaction, and randomly combined in pCANTAB-5E vector. The resulting human scFv phage display library had $3{\times}10^4$ independent clones, and biopanning was performed with house dust mite extracts. Results: Four scFv clones specific for house dust mite extract were isolated. Immunoblot assay showed that our clones reacted to 25 kDa and 50~60 kDa proteins with unknown identity in mite extracts. Sequence analysis indicated that two clones (b7 and c15) are identical, and all clones belong to human $V_H3$ subgroup. On the other hand, light chain usage was different in that two clones (a2 and b7 / c15) belonging to V ${\kappa}4$ subgroup, but a4 used V ${\kappa}1$ light chain gene. Conclusion: Our approach should facilitate provision of useful information on the antibody responses against allergens at the molecular level in humans.

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.319-321
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• 2005

본 연구진은 phage display peptide library로부터 $TiO_{2}$ nanoparticle에 binding 하는 peptide를 선별하여 보고한바 있다. 이 중의 하나인 PEP9을 선택하여 alanine scanning mutagenesis를 통하여 mutant peptide를 display하는 phage를 제작하여 $TiO_{2}$에의 binding을 조사하였다. 그 결과, 4번 위치의 proline이 alanine으로 치환된 peptide의 경우 binding activity가 $10\%$로 감소하였고, 2번 valine, 3번 serine, 5번 isoleucine의 치환 peptide는 binding이 $40\%$로 감소하였다. 이러한 사실로 미루어볼 때, PEP9과 $TiO_{2}$ nanoparticle의 결합에는 2, 3, 4, 5번의 아미노산이 만들어내는 3차원적 구조가 중요한 역할을 하는 것으로 결론 내릴 수 있었다.

• 한국축산식품학회지
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• 제31권1호
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• pp.47-53
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• 2011

Salmonella enterica serovar Typhimurium (ST) is one of the most common serovars isolated from humans and animals. It has been suggested that ST infections in Koreans are largely due to the consumption of contaminated pork and beef. To investigate the genotypes, phage types, and antimicrobial resistance patterns for ST isolates of different origins, a total of 70 ST strains, including 19 isolates from humans, 44 isolates from pigs, and 6 isolates from cattle, were analyzed using pulsedfield gel electrophoresis (PFGE), phage typing, and antimicrobial susceptibility tests. Forty-three distinct PFGE patterns were generated from 70 ST isolates, which were grouped into 14 PFGE groups (from A to N) at the level of 75% similarity. The most prevalent group was the A (A1-A17 subtypes) group, encompassing 54.5% (38/70) of ST isolates. ST isolates from pigs and cattle mostly belong to groups A and L, whereas ST isolates from humans mostly belong to groups F and C. Antimicrobial susceptibility tests using 11 antimicrobial agents showed that resistance to tetracycline (TE) (81.4%) was highly prevalent, followed by streptomycin (S) (64.3%) and nalidixic acid (NA) (31.4%) resistance. A total of seventeen antimicrobial resistance patterns were observed. Only 8.6% of isolates, including a reference strain, were susceptible to all antimicrobial agents tested. The most prevalent resistance pattern was TE-S (37.1%), which was seen in 66.6% of bovine, 40.8% of swine and 21.1% of human isolates. Three ST isolates from humans (15.9%) showed resistance to 7-8 antimicrobials. The most predominant phage type (PT) was U302 (64.3%), followed by DT170 (10.0%). PFGE types did not coincide with antimicrobial resistance patterns and phage types; therefore, the combination of those types allowed for further differentiation between tested ST isolates.

• 대한수의학회지
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• 제40권3호
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• pp.505-514
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• 2000

This study was conducted to investigate the isolation prequency, serotypes, and related epidemiological properties of 341 Salmonella spp from domestic poultry and environmental samples during the period 1993-1995. A total of 1,918 samples was collected during the three years period in nationwide. Most of Salmonella spp were isolated from the intestinal contents of poultry, especially cecal(46.0%) and rectal(35.8%) contents. Among the tested samples, rat(28.5 %) was the most predominant Salmonella reservoirs and followed by duck(24.8%), broiler(18.8%), layer(14.8%) and feed(7.1%), in order. More than twelve Salmonella serovars were identified among the 341 Salmonella isolates. The most prevalent serotypes isolated from non-human sources were S enteritidis (22.3%) and S pullorum (21.9%), S muenchen (13.9%), S typhimurium (12.6%), S gallinarum, S meleagridis, S heidelberg, and S senftenberg were followed, in order. In layer chickens, S pullorum (26.0%) was the most predominant serotype but S muenchen (33.0%) was in broiler chickens, S enteritidis (28.4%) was in ducks, and S typhimurium (60.0%) was in rats, respectively. As a results, S enteritidis was identified as the most prevalent serotype in non-human Salmonella isolates in Korea during the period 1993-1995. A preliminary study on the phage typing of 19 S enteritidis selected from the nationwide scale was shown that S enteritidis phage type(PT) 4 was the most predominant PT, and SEPT 1, SEPT 6a, SEPT 7 and SEPT 7a variant were also found in the same period.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.122-128
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• 1992

Runaway replciation plasmid pSY35AT를 이용하여 이 plasmid가 가지고 있는 $cI_{857}$ repressor를 숙주 MC1065를 이용하여 $30^{\circ}C$에서 $37^{\circ}C$로 온도를 올려 shift-up 법으로 생산하였다. $cI_{857}$은 wild type cI repressor 정제법을 수정하여 사용하였으며 정제된 $cI_{857}$ repressor 농도는 0.11mg/ml이었다. 그리고 정제된 $cI_{857}$ repressor와 $^3H-CTP$로 labelinf한 PrOr과의 결합활성은 cell 파쇄액보다 약 23배 높았으며, 온도가 올라갈수록 결합활성은 떨어졌다.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.38-43
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• 2016

Novel and specific recognition elements are of central importance in the development of a pathogen detection method. Here, we describe a simple method for identifying the cell-wall binding domain (CBD) from a sequenced bacterial genome employing homology search for phage lysin genes. A putative CBD (CPF369_CBD) was identified from a genome of Clostridium perfringens type strain ATCC 13124, and its function was studied with the CBD-GFP fusion protein recombinantly expressed in Escherichia coli. Fluorescence microscopy showed the specific binding of the fusion protein to C. perfringens cells, which demonstrates the potential of this method for the identification of novel bioprobes for specific detection of pathogenic bacteria.

• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.592-596
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• 2014

HMO-2011, a recently isolated lytic phage that infects the SAR116 bacterial clade, represents one of the most abundant phage types in the oceans. In this study, the HMO-2011 genome sequence was compared with virome sequences obtained from various depths of the Pacific Ocean regions using metagenome binning. HMO-2011 was confirmed to be one of the most highly assigned viruses, with a maximum of 7.6% of total reads assigned. The HMO-2011-type phages demonstrated a depth-specific distribution, showing more abundance in the euphotic zone of coastal, transition, and open ocean regions as compared with the dark ocean.