Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan.
Biomolecules. 2021 May 29;11(6):810. doi: 10.3390/biom11060810.
In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR V-7D12, in a thermally denatured state at a high temperature. We produced V-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the expression of V-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all V-7D12 variants. Moreover, all V-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where V-7D12 variants are unfolded, showed that the untagged V-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.
在这项研究中，我们评估了精氨酸和赖氨酸可溶性增强肽（SEP）标签控制模型蛋白抗 EGFR V-7D12 在高温下热变性状态下的可溶性的潜力。我们生产了带有 C 端 SEP 标签的 V-7D12 抗体，该标签由五个或九个精氨酸或赖氨酸组成（分别为 7D12-C5R、7D12-C9R、7D12-C5K 和 7D12-C9K）。5-精氨酸和 5-赖氨酸 SEP 标签使 V-7D12 的表达增加了 80%以上。生物物理和生化分析证实了所有 V-7D12 变体的天然类似二级和三级结构特性以及单体性质。此外，所有 V-7D12 变体均保留了与 EGFR 细胞外结构域的完全结合活性。最后，在 60 和 75°C 下孵育 45 分钟的热应激下，V-7D12 变体处于展开状态，结果表明未标记的 V-7D12 在所有四种缓冲液中均形成聚集体，上清液蛋白浓度降低了高达 35%。7D12-C5R 和 7D12-C9R 未在乙酸钠（pH4.7）和 Tris-HCl（pH8.5）中聚集，但在磷酸盐缓冲液（pH7.4）和磷酸盐缓冲盐水（pH7.4）中形成聚集体。赖氨酸标签（无论是 C5K 还是 C9K）都具有最强的溶解作用，并且 7D12-C5K 和 7D12-C9K 均留在上清液中。总的来说，我们的结果表明，在热应激条件下，赖氨酸 SEP 标签的溶解效果比精氨酸 SEP 标签更强，并且 SEP 标签不会影响蛋白质的结构和功能特性。
