Centre for Immune Regulation, University of Oslo, Oslo, Norway.
BMC Biotechnol. 2010 Feb 3;10:8. doi: 10.1186/1472-6750-10-8.
Efficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs.
The effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Valphabeta domain orientation was far superior to the Vbetaalpha domain orientation regarding monomeric yield of functionally folded molecules.
The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.
抗体（Ab）分子的高效表达系统已经存在，这使得对大量个体 Ab 变体进行特征分析成为可能。相比之下，可溶性 T 细胞受体（TCR）的表达系统尚未建立。试图构建细菌系统通常会导致产量低，且所得到的材料容易聚集和发生蛋白水解。在此，我们提出了一种优化的周质细菌表达系统，用于可溶性单链（sc）TCR。
研究了 1）过表达周质分子伴侣 FkpA，2）培养条件和 3）分子设计的影响。FkpA 的高水平表达可以防止过早聚集和包涵体形成，从而有助于周质可溶性 scTCR 的表达。周质表达可以促进二硫键的形成，这是 scTCR 达到正确折叠状态的前提。它还可以快速、轻松地回收正确折叠的蛋白质，而无需耗时的下游处理。不使用 IPTG 诱导进一步提高了周质表达的产量，而在生长培养基中添加蔗糖对产量的影响很小。通过摇瓶培养，获得了毫克级的具有活性的纯化产物。与 Vbetaalpha 结构域取向相比，Valphabeta 结构域取向在功能折叠分子的单体产量方面具有显著优势。
本文提出的通用表达方案允许快速生产可溶性 scTCR，适用于 1）高产量回收，足以进行生物物理特性分析，以及 2）对这些分子进行高通量筛选，进行分子工程改造。
