Abstract
Covalent modification of a protein with polyethylene glycol (PEG) has become one of the most widely used and well established drug enhancement strategies in the biopharmaceutical industry. The general benefits enjoyed by PEGylation, such as prolonged serum half-lives or reduced immunogenicity in vivo, are well known. By now the PEGylation process has been performed with purified proteins, and it is required to recover the desired PEGylate by a multi-step purification process. The ultimate aim of our research is to develop an integrated process of PEGylation and in vitro refolding starting with inclusion body material. For this, we investigated the feasibility that a protein could be PEGylated under a denaturing condition and also the PEGylated proteins could be refolded correctly. Using lipase as a model protein, we found that it was PEGylated in the presence of 8M urea and that the PEG molecules covalently attached to lipase did not appear to hinder its refolding.
변성제 (urea)와 환원제에 의해 완전히 풀린 상태의 lipase도 PEG에 의해 수식되는 것을 관찰하였다. 또한 mPEG-aldehyde로 수식된 mono-PEGylate과 di-PEGylate을 변성제와 환원제를 이용해 unfolding 시킨 후 희석에 의한 재접힘 시킨 결과, lipase에 공유결합된 PEG 분자는 재접힘 수율에 거의 영향을 미치지 않는 것으로 나타났다. 따라서 내포체 단백질을 대상으로 변성된 상태에서 PEGylation시킨 후 in vitro 재접힘 공정을 통해 PEGylation된 상태의 재생된 단백질을 회수할 수 있는 통합공정의 타당성을 제시하였다.
