Nekoufar Samira, Fazeli Ahmad, Fazeli Mohammad Reza
Department of Biochemistry, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Adv Pharm Bull. 2020 Jun;10(2):233-238. doi: 10.34172/apb.2020.027. Epub 2020 Feb 18.
Solubilization of inclusion bodies expressed in is a critical step during manufacturing of recombinant proteins expressed as inclusion bodies. So far, various methods have been used for solubilization and purification of inclusion body proteins to obtain active proteins with high purity and yield. The aim of this study was to examine the benefit of organic solvents such as alcohols in solubilization of recombinant interferon β-1b inclusion bodies. Effect of important parameters inclusion pH, concentration and type of denaturant and concentration of alcoholic solvents were optimized to formulate a suitable solubilization buffer and investigate their effect on solubilization of interferon β-1b inclusion bodies. Our findings showed the acidic pH in the range of 2-3 is more suitable than alkaline pH >12 for solubilization and achieving higher content of interferon β-1beta and pure recombinant protein. We have also demonstrated that 1% SDS acts better than 2M urea to solubilize Inclusion body proteins of interferon β-1b at pH of 2-3. The interferon concentration was 2.35 mg per 100 mg IB when we used 40% () 1-propanol and 20% () 2-butanol into the buffer solution as well. The optimized method provides gentile condition for solubilization of inclusion body at high protein concentration and purity with a degree of retention of native secondary structure which makes this method valuable to be used in production and research area.
在重组蛋白以包涵体形式表达的生产过程中,包涵体的溶解是关键步骤。到目前为止,已采用多种方法来溶解和纯化包涵体蛋白,以获得高纯度和高产量的活性蛋白。本研究旨在探讨醇类等有机溶剂在重组干扰素β-1b包涵体溶解中的作用。优化了包括pH值、变性剂浓度和类型以及醇类溶剂浓度等重要参数的影响,以配制合适的溶解缓冲液,并研究它们对干扰素β-1b包涵体溶解的影响。我们的研究结果表明,pH值在2 - 3范围内的酸性条件比pH值>12的碱性条件更适合溶解,并且能获得更高含量的干扰素β-1β和纯重组蛋白。我们还证明,在pH值为2 - 3时,1% SDS比2M尿素在溶解干扰素β-1b包涵体蛋白方面效果更好。当我们在缓冲溶液中使用40%(体积分数)1-丙醇和20%(体积分数)2-丁醇时,每100 mg包涵体中的干扰素浓度为2.35 mg。该优化方法为在高蛋白浓度和纯度下溶解包涵体提供了温和的条件,并保留了一定程度的天然二级结构,这使得该方法在生产和研究领域具有应用价值。