Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Mol Biotechnol. 2021 Jan;63(1):80-89. doi: 10.1007/s12033-020-00286-4. Epub 2020 Nov 9.
Pichia pastoris expression system was introduced with post-translation process similar to higher eukaryotes. Preliminary studies were performed toward process intensification and magnetic immobilization of this system. In this experiment, effects of magnetic immobilization on the structure of recombinant protein were evaluated. P. pastoris cell which express human serum albumin (HSA) was used as a model. The cells were immobilized with various concentrations of APTES coated magnetite nanoparticles. HSA production was done over 5 days induction and structure of the product was analyzed by UV-vis, fluorescence, and ATR-FTIR spectroscopy. Second derivative deconvolution method was used to analyze the secondary structure of HSA. P. pastoris cell that were immobilized with 0.5 and 1 mg/mL of nanoparticles were produced HSA with intact structure. But immobilization with 2 mg/mL of nanoparticles resulted in some modifications in the secondary structures (i.e., α-helixes and β-turns) of produced HSA. Based on these data, immobilization of P. pastoris cells with 0.5 or 1 mg/mL of nanoparticles is completely efficient for cell harvesting and has any effect on the structure of recombinant product. These findings revealed that decoration of microbial cells with high concentrations of nanoparticles has some impacts on the structure of secretory proteins.
毕赤酵母表达系统具有类似于高等真核生物的翻译后加工过程。本研究初步探讨了该系统的强化和磁性固定化。在本实验中，评估了磁性固定化对重组蛋白结构的影响。使用表达人血清白蛋白（HSA）的毕赤酵母细胞作为模型。用不同浓度的 APTES 修饰的磁铁矿纳米粒子固定化细胞。诱导表达 5 天后进行 HSA 生产，并通过紫外-可见光谱、荧光光谱和衰减全反射傅里叶变换红外光谱（ATR-FTIR）分析产物的结构。使用二阶导数去卷积法分析 HSA 的二级结构。用 0.5 和 1 mg/mL 纳米粒子固定化的毕赤酵母细胞产生的 HSA 具有完整的结构。然而，用 2 mg/mL 纳米粒子固定化会导致产生的 HSA 的二级结构（即α-螺旋和β-转角）发生一些修饰。基于这些数据，用 0.5 或 1 mg/mL 纳米粒子固定化毕赤酵母细胞对细胞收获完全有效，并且对重组产物的结构没有影响。这些发现表明，用高浓度的纳米粒子修饰微生物细胞会对分泌蛋白的结构产生一些影响。
