Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Expression Properties and Skin Permeability of Human Basic Fibroblast Growth Factor with or without PTD Fused to N- or C-terminus in Escherichia coli

대장균 발현시스템에서 단백질 전달 도메인 PTD가 인간 섬유아세포 성장인자(FGF2)의 N- 또는 C-말단에 결합 되었을 때 미치는 재조합 단백질 복합체의 발현 특성과 피부 투과능력

  • Received : 2017.11.17
  • Accepted : 2018.03.22
  • Published : 2018.03.30
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Abstract

Human fibroblast growth factor (FGF) has the potential to be a commercially important therapeutic or cosmeceutical agent due to its ability to generate tissue and heal wounds. Granting permeability into skin tissues increases the therapeutic effects of FGF. Thus, several researchers have attempted the fusion of FGF conjugates with protein transduction domains (PTDs) to investigate the transduction ability and therapeutic effects of FGF. Less is known, however, about whether the location of PTD fused to the N- or C-terminus of FGF proteins has a significant impact on the folding and stability in Escherichia coli, and eventually, on transduction. Here, we report cloning of human basic fibroblast growth factor (FGF2) as a control and FGF2 with PTD fused to the N- or C-terminal ends of FGF proteins by an overlap extension PCR. We performed expression, verified expression properties of recombinant FGF2 without or with PTD fused to the N-terminus and the C-terminus, and investigated transduction ability into tissue by treating the dorsal skin of mice subjects. As a result, FGF2 and FGF2-PTD (fused to C-terminus) fusion protein were expressed as soluble forms suitable for straight-forward purification, unlike insoluble PTD-FGF2 (fused to N-terminus), but only FGF2-PTD fusion protein could transduce into the dorsal skin tissue of the mice subjects. Our results suggest that FGF2 with PTD fused to the C-terminus is more efficient than other options in terms of expression, purification, and delivery into skin tissue, as it does not require labor-intensive, costly, and time-consuming methods.

인간 섬유아세포 성장인자는 조직 생성 및 상처 치료 효과로 인해 상업적으로 중요한 치료제 또는 화장품소재로서 가능성이 높다. 피부 조직에 침투성을 부여하여 치료효과를 높이기 위해서 단백질 전달 도메인인 PTD를 FGF에 융합을 시도하고 있으며 피부로의 투과능력과 그로인한 치료 효과에 대한 연구도 진행되고 있다. 그러나, PTD가 FGF 단백질의 N- 또는 C-말단에 결합 되었을 때 PTD의 위치가 대장균 발현시스템에서 재조합단백질 접힘 및 안정성, 그리고 결국 피부로의 도입능력에 상당한 영향을 미치는지에 대해서는 알려져 있지 않다. 여기에서 우리는 대조군으로 PTD가 융합되지 않은 인간 염기성 섬유아세포 성장인자(FGF2)와 PTD가 FGF2의 N-말단 또는 C-말단에 융합된 FGF2 복합체를 중합효소연쇄반응(OE-PCR)을 통해 클로닝 하였다. 그 다음 이들 재조합 FGF2의 단백질 발현 및 특성을 확인하고 마우스 등 피부를 이용하여 조직 내로의 도입 능력을 조사 하였다. 결과적으로, 불용성 PTD-FGF2 (N 말단 융합)와는 달리 대조군 FGF2와 FGF2-PTD 융합 단백질(C- 말단 융합)은 가용성 형태로 발현되어 재조합단백질 획득이 용이하였고, 마우스 피부 도입능력은 FGF2-PTD 융합단백질에서만 나타내 보였다. 우리의 결과는 C-말단에 융합된 FGF2-PTD 융합단백질이 발현, 정제, 피부 투과능력의 측면에서 다른 두 옵션들보다 노동, 비용, 시간면에서 보다 더 효율적일 수 있음을 시사한다.

Keywords

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