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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Purification of Recombinant CTP-Conjugated Human prostatic acid phosphatase for activation of Dendritic Cell

수지상세포 활성화를 위한 세포투과 펩타이드가 결합된 재조합 전립성 산성 인산분해효소의 정제

  • Yi, Ki-Wan (Protein Engineering Team, CreaGene Inc.) ;
  • Ryu, Kang (Protein Engineering Team, CreaGene Inc.)
  • 이기완 (크레아젠(주) 단백질 공학팀) ;
  • 류강 (크레아젠(주) 단백질 공학팀)
  • Published : 2009.02.28
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Abstract

Human prostatic acid phosphatase (PAP), with comprehensive homology to glandular kallikrein, are representative serum biomarkers of prostate cancer. Dendritic cell (DC), which is the potent antigen-presenting cells(APC) in the immune system, can induce strong T cell responses against viruses, microbial pathogens, and tumors. Therefore, the immunization using DC loaded with tumor-associated antigens is a powerful method for inducing anti-tumor immunity. The CTP (Cytoplasmic Transduction Peptide) technology developed by Creagene which can transport attached bio-polymers like nucleic acids or proteins into the cell with high permeation efficiency. As the active forms of PAP can mediate apoptotic processing, we used multimer forms of PAP as an inactive form for antigen pulsing of DCs. In this study, multimeric forms of CTP-rhPAP was obtained according to the advanced purification process and subsequently confirmed by gel filtration chromatography, western blot and Dynamic Light Scattering. Therefore, CTP-conjugated PA multimers transduced into the cytoplasm were efficiently presented on the cell surface without any harm effect on cells via MHC class I molecules and result in induction of a large number of effector cell.

glandular kallikrein에 광범위한 상동성을 가지는 인간 전립성 산성 인산 가수분해 효소는, 전립선암의 대표적인 혈청 biomarkers이다. 수지상세포는 유력한 항원 제시 세포이며, 바이러스, 미생물 병원체 및 종양에 대하여 면역 계통에서 강력한 T 세포 응답을 유도할 수 있다. 따라서, 종양 특이적인 항원으로 감작된 수지상세포를 이용한 면역요법은 anti-tumor 면역 유도를 위한 강력한 방법중의 하나이다. 크레아젠(주)에서 개발된 CTP (세포막 투과성 펩티드) 기술은 세포 내로의 높은 침투 효율성을 가지며 핵산이나 단백질과 같은 생체 고분자 물질을 접합하여 세포내로 수송할 수 있는 기술이다(36). 하지만 활성형의 인간 전립성 산성 인산 가수분해 효소는 세포사멸을 매개할 수 있기 때문에, 본 연구진은 항암 치료용 백신으로의 수지상세포 감작을 위해 비활성형 형태의 다중체 (multimer) 항원을 개발하였다. 본 연구에서, 수지상 세포의 감작과 활성화에 안전하고 효율적인 다중체 형태 (multimeric form)의 세포막 투과성 펩티드가 융합된 인간 전립성 산성 인산 가수분해 효소를 얻기 위한 정제공정을 기법을 개발하였고 젤 여과 크로마토그래피, western blot과 Dynamic Light Scattering을 이용하여 확인하였다. 아울러, 정제된 다중체 형태 (multimeric form)의 세포막 투과성 펩티드가 융합된 인간 전립성 산성 인산 가수분해 효소는 수지상 세포의 감작시 세포질 내로 효과적으로 침투되었다. 결과적으로 세포의 사멸의 부작용이 없이 MHC class I 분자를 통해 수지상세포의 표면으로 효과적으로 제시되었다.

Keywords

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