대장균의 alkaline phosphatase가 융합된 anti-DR4 single-chain Fv (ScFv) 항체의 개발 = The development of anti-DR4 single-chain Fv (ScFv) antibody fused to Escherichia coli alkaline phosphatase

국문 초록 (Abstract)

항체의 특이적 결합을 분석하는 효소면역분석법은 항원의 탐지를 위해 주로 horseradish peroxidase (HRP) 또는 alkaline phosphatase (AP) 등의 효소를 사용한다. 이때 효소를 주로 화학적으로 항체에 결합시켜 사용하게 되는데 이 과정이 복잡하며 불규칙하게 일어나서 항체 및 효소의 기능을 감소시키게 된다. 또한 대부분의 효소면역분석법에서는 주로 일차 항체의 항원결합을 탐지하기위해 이차 항체를 사용하는데, 즉 이차 항체에 결합한 효소의 기질발색에 의해 일차 항체의 항원결합을 탐지하므로 이차 항체가 요구 되어질 뿐만 아니라 이차 항체의 일차 항체에 대한 반응을 위한 부가적인 배양시간이 필요하다. 더욱 더 중요한 것은 이차 항체만의 비특이적 항원 결합 역시 제거되어져야 한다. 본 연구에서는 대장균의 genomic DNA로부터 PCR을 통해 alkaline phosphatase 유전자(Sadeghi et al., 2008)를 증폭 분리한 다음 이를 TRAIL (tumor necrosis factor ${\alpha}$ related apoptosis induced ligand) receptor인 death receptor 4 (DR4)에 특이적으로 결합하는 hAY4 single-chain Fv (ScFv)에 융합시킨 재조합 ScFv-AP 형태로 대장균에서 발현시켜 정제하였다. 정제된 hAY4 ScFv-AP는 SDS-PAGE에서 단량체(monomer) 분자량인 73.8 kDa을 나타내었다. 그러나 size-exclusion chromatography(SEC)에서는 147.6 kDa을 나타내는 결과를 통해 hAY4 ScFv-AP는 AP의 자연적인 비공유결합에 의해 이량체(dimeric form)형성이 유도되어짐을 확인하였다. 또한 ELISA, Western blot 그리고 immunocytochemistry에서 이차 항체 없이 일차 항체 hAY4 ScFv에 직접 융합된 AP의 기질발색에 의해 ScFv 일차 항체의 특이적 항원결합을 나타내었다. 요약하면 hAY4 ScFv와 대장균의 alkaline phosphatase 유전자를 융합시켜 대장균에서 수용성 형태로 성공적으로 정제하였으며 정제된 ScFv-AP 융합단백질은 ELISA, Western blot 및 immunocytochemistry에서 항원결합력을 나타냈으며 또한 구매에 따른 고비용, 부가적인 배양시간 및 비특이적 결합에 의한 오류 등의 문제점을 갖는 이차 항체를 사용하지 않고 직접적인 항원결합력을 나타내었다.

다국어 초록 (Multilingual Abstract)

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

참고문헌 (Reference)

1 Coleman, J. E, "Structure and mechanism of alkaline phosphatase" 21 : 441-483, 1992

2 Muller, B. H., "Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization" 227 : 177-185, 1999

3 Ducancel, F., "Recombinant colorimetric antibodies:construction and characterization of a bifunctional f (ab)2/alkaline phosphatase conjugate produced in Escherichia coli" 11 : 601-605, 1993

4 Carrier, A., "Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human iggs: Application to anti-hbsag detection" 181 : 177-186, 1995

5 Liu, X., "Production and characterization of a singlechain Fv antibody–alkaline phosphatase fusion protein specific for clenbuterol" 45 : 56-64, 2010

6 Volkel, T., "Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies" 14 : 815-823, 2001

7 Trinh, R., "Optimization of codon pair use within the (ggggs)3 linker sequence results in enhanced protein expression" 40 : 717-722, 2004

8 Sadeghi, H. M. M., "Optimization of alkaline phosphatase gene expression in E. coli" 3 : 35-39, 2008

9 Mandecki, W., "Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat" 4 : 801-804, 1991

10 Solar, I., "Linker modification introduces useful molecular instability in a single chain antibody" 8 : 717-723, 1995

11 Duenas, M., "Intra-and extracellular expression of an scFv antibody fragment in E. coli: effect of bacterial strains and pathway engineering using GroES/L chaperonins" 16 : 476-477, 1994

12 Acchione, M., "Impact of linker and conjugation chemistry on antigen binding, fc receptor binding and thermal stability of model antibody-drug conjugates" 4 : 362-372, 2012

13 Kim, J. K., "Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis" 24 : 542-548, 1994

14 Lee, S. H., "Humanization of an agonistic anti-death receptor 4single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity" 47 : 816-824, 2010

15 Le Gall, F., "Effect of linker sequences between the antibody variable domains on the formation, stability and biological activity of a bispecific tandem diabody" 17 : 357-366, 2004

16 Park, K. J., "Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen" 27 : 289-295, 2005

17 Suzuki, C., "Construction, bacterial expression, and characterization of hapten-specific single-chain fv and alkaline phosphatase fusion protein" 122 : 322-329, 1997

18 Wels, W., "Construction, bacterial expression and characterization of a bifunctional single-chain antibody-phosphatase fusion protein targeted to the human erbb-2 receptor" 10 : 1128-1132, 1992

19 Wang, S., "Construction of multiform scFv antibodies using linker peptide" 35 : 313-316, 2008

20 Lei, S. P., "Characterization of the Erwinia carotovora pelB gene and its product pectate lyase" 169 : 4379-4383, 1987

21 Harlow, E., "Antibodies: A lab manual" Cold Spring Harbor Laboratory Press 1988

22 Whitlow, M., "An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability" 6 : 989-995, 1993

23 Wang, C. L., "A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity" 7 : 715-722, 1994

24 Hu, Z. Q., "A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains" 764 : 84-92, 2013