Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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The development of anti-DR4 single-chain Fv (ScFv) antibody fused to Streptavidin

Streptavidin이 융합된 DR4 항원에 특이적인 single-chain Fv 항체의 개발

  • Kim, Seo Woo (Division of Biohealth Science, College of Natural Sciences, Changwon National University) ;
  • Wu, Sangwook (Department of Physics, College of Natural Sciences, Pukyong National University) ;
  • Kim, Jin-Kyoo (Division of Biohealth Science, College of Natural Sciences, Changwon National University)
  • 김서우 (창원대학교 자연과학대학 생명보건학부) ;
  • 우상욱 (부경대학교 자연과학대학 물리학과) ;
  • 김진규 (창원대학교 자연과학대학 생명보건학부)
  • Received : 2018.09.05
  • Accepted : 2018.10.09
  • Published : 2018.12.31
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Abstract

The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.

Streptavidin (STR)과 Biotin system은 Biotin의 Streptavidin에 대한 높은 비공유 친화력(non-covalent affinity; $K_D=10^{-14}M$)과 4 Biotin 결합부위를 갖는 Streptavidin의 tetramer 구조로 인해 복수의 항원결합부위 및 복수의 항원특이성을 갖는 항체를 제조할 수 있기 때문에 가장 활발하게 연구되고 있다. 이 system을 활용하기 위해 우리는 Streptomyces avidinii 염색체 DNA로부터 PCR을 통해 Streptavidin (STR) 유전자를 증폭하고 이를 TRAIL (tumor necrosis factor ${\alpha}$ related apoptosis induced ligand) receptor인 death receptor 4 (DR4)에 특이적으로 결합하는 hAY4 single-chain Fv 항체유전자에 융합시켰다. 대장균에서 발현시킨 STR에 융합된 hAY4 ScFv (hAY4-STR) 항체는 가열시킨 SDS-PAGE에서 43 kDa monomer를 나타내었다. 그러나 가열하지 않은 SDS-PAGE와 Size-exclusion chromatography에서는 tetramer인 172 kDa을 나타내었는데 이는 hAY4 ScFv-STR 항체가 STR의 자연적인 비공유결합에 의해 유도된 tetramer를 형성하고 있음을 나타내고 있다. 본 융합 단백질은 Ouchterlony assay와 ELISA에서 보여주는 것처럼 자연 Streptavidin과 유사한 Biotin 결합력을 유지하고 있었다. ELISA와 Westernblot을 이용하여 정제된 hAY4-STR 융합항체의 DR4 항원결합력 또한 확인하였다. 게다가 표면 플라즈몬 공명(surface plasmon resonance) 분석에서 hAY4 ScFv-STR tetramer는 tetramerization에 의해 hAY4 ScFv monomer보다 60배 더 높은 항원결합력을 나타내었다. 요약하면 hAY4 ScFv-STR 융합단백질은 E. coli에서 soluble tetramer로 성공적으로 발현 및 정제되었으며 Biotin과 DR4 항원에 동시에 결합함을 보여 주었다. 이는 bifunctional and tetrameric ScFv 항체를 제조 할 수 있음을 제시해 주고 있다.

Keywords

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Fig. 1. DNA gel analysis of DR4, Streptavidin, hAY4 ScFv and hAY4 ScFv-STR genes cloned into pUC 119 expression vector.

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Fig. 2. Plasmid construction of Streptavidin, DR4, hAY4 ScFv and hAY4 hAY4-STR.

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Fig. 3. SDS-PAGE analysis of soluble proteins expressed in E. coli.

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Fig. 4. SDS-PAGE analysis of Streptavidin and hAY4 ScFv-STR using heating or non-heating condition to determine non-covalent tetramerization effect.

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Fig. 5. Size exclusion chromatography (SEC).

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Fig. 6. Ouchterlony assay and ELISA analysis to determine Biotin binding activity of Streptavidin and hAY4 ScFv-STR.

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Fig. 7. ELISA analysis and Westernblot to determine antigen binding activity of hAY4 ScFv-STR.

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Fig. 8. Surface plasmon resonance (SPR) analysis of hAY4 ScFv, STR and hAY4 ScFv-STR.

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Fig. 9. 3D modeling of hAY4 ScFv-STR tetramer.

Table 1. Primers used for PCR amplification of DR4, Streptavidin, hAY4 ScFv, and hAY4 ScFv-Streptavidin genes

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Table 2. Kinetic binding parameters for the interactions of hAY4 ScFv and hAY4 ScFv-STR with DR4 determined by SPR analysis

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