VTT Technical Research Centre of Finland, Oulu, Finland.
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
Sci Rep. 2020 Feb 12;10(1):2472. doi: 10.1038/s41598-020-59091-3.
Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.
时间门控表面增强拉曼光谱（TG-SERS）被用于评估大肠杆菌中重组蛋白的生产。TG-SERS 抑制了细菌和培养基中生物分子的荧光信号。在细胞培养的不同时间点观察到了特征性的蛋白质特征，并与传统的连续波（CW）-Raman 与 SERS 进行了比较。TG-SERS 可以区分蛋白质的离散特征，例如二级结构，因此可以指示蛋白质的折叠或展开。首次使用纳米原纤维纤维素作为纳米颗粒和细菌细胞的稳定剂的新方法被用于增强拉曼信号，同时抑制背景信号。我们使用分批发酵模式评估了复杂培养基中 hCNTF、hHspA1 和 hHsp27 的表达。还使用 EnBase 以类似分批补料的方式培养了 HspA1。由于细胞内的聚集问题，HspA1 的表达效果不佳，而在批式培养中表达的 hCNTF 则正确折叠，并在 EnBase 培养中鉴定出蛋白质不稳定性。时间门控拉曼光谱被证明是一种强大的工具，可以在培养过程中评估活大肠杆菌细胞中的蛋白质生产和正确折叠。
