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Streptavidin이 융합된 GFP항원 특이적인 VHH 항체의 기능적 발현

Functional Expression of an Anti-GFP Camel Heavy Chain Antibody Fused to Streptavidin

  • 한승희 (창원대학교 자연과학대학 생명보건학부) ;
  • 김진규 (창원대학교 자연과학대학 생명보건학부)
  • Han, Seung Hee (Division of Biohealth Science, College of Natural Sciences, Changwon National University) ;
  • Kim, Jin-Kyoo (Division of Biohealth Science, College of Natural Sciences, Changwon National University)
  • 투고 : 2018.09.17
  • 심사 : 2018.10.25
  • 발행 : 2018.12.30

초록

Biotin에 강한 결합 친화력($K_D=10^{-14}M$)과 함께 streptavidin의 tetramer 특징은 VHH 항체를 streptavidin에 융합시키게 하여 biotinylated horseradish peroxidase를 사용하는ELISA 와Western blot analysis 등의 면역분석법에서 VHH 항체의 항원결합력을 증가시키는데 응용 가능하다. 이를 응용하기 위해 우리는 Streptomyces avidinii 염색체 DNA로부터 PCR을 통해 streptavidin유전자를 증폭하고 이를 green fluorescent protein항원에 특이적으로 결합하는 8B9 VHH 항체유전자에 융합시켰다. 대장균에서 수용성 융합단백질로 발현시키기 위해 pUC119 플라스미드에 기초한 발현시스템을 사용하였다. 즉 lacZ promoter를 사용하여 IPTG에 의해 단백질발현을 유도하게 하였으며, 아미노말단에 pelB leader를 두어 발현된 단백질의 periplasmic space로 이동하게 하여 수용성 단백질형태의 분비를 촉진하였으며 카르복시말단에 6개의 polyhistidine tags를 두어 $Ni^+$-NTA-agarose column을 사용하여 발현된 단백질을 정제하였다. Streptavidin이 biotin에 강하게 결합함으로 대장균에 독성을 나타냄에도 불구하고 본 수용성 융합단백질은 성공적으로 발현되었다. SDS-PAGE에서 가열하는 경우 변성되어 30.6 kDa를, 가열하지 않는 경우에는 자연 상태의 122.4 kDa을 나타내었다. 이는 8B9 VHH항체에 융합된 streptavidin moiety에 의해 monomer subunit가 비공유결합으로 tetramerization됨을 제시해준다. 또한 본 융합단백질은 ELISA와 Westernblot analysis에서 보여진 것처럼 parental streptavidin과 유사한 biotin결합력과 green fluorescent protein항원 결합력을 모두 나타내었다. 결론적으로 streptavidin에 융합된 8B9 VHH 항체형태의 융합단백질은 대장균에서 수용성 tetramer로 성공적으로 발현 및 정제되었으며 biotin과 green fluorescent protein 항원에 동시에 결합함으로써 tetrameric and bifunctional VHH 항체제조의 가능성을 제시해주었다.

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

키워드

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Fig. 1. DNA gel analysis of Streptavidin, GFP, 8B9 VHH and 8B9 VHH-STR genes cloned into pUC 119 expression vector.

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Fig. 2. Plasmid construction of streptavidin, GFP, 8B9 VHH and 8B9 VHH-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 and Westernblot analysis of 8B9 VHHSTR expressed in E. coli.

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Fig. 5. ELISA analysis and Westernblot analysis to determine biotin binding activity of 8B9 VHH-STR.

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Fig. 6. ELISA analysis and Westernblot analysis to determine GFP antigen binding activity of 8B9 VHH-STR.

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Fig. 7. 3D modeling of 8B9 VHH-STR tetramer.

Table 1. Primers used for PCR amplification of GFP, Streptavidin, 8B9 VHH and 8B9 VHH-Streptavidin genes.

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Table 2. Biochemical analysis of GFP, Streptavidin, 8B9 VHH and 8B9 VHH-STR

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