Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA
Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA
复制标题
DOI：
10.1186/1472-6750-10-8
复制
发表时间：
2010-02-03
影响因子：
3.5
通讯作者：
Loset, Geir A.
中科院分区：
文献类型：
--
作者：
Gunnarsen, Kristin S.;Lunde, Elin;Loset, Geir A.
背景资料：存在用于抗体（Ab）分子的有效表达系统，其允许表征大量单个Ab变体。相反，这种表达系统缺乏可溶性T细胞受体（TCR）。产生细菌系统的尝试通常导致低产率和易于聚集和蛋白水解的材料。结果：研究了周质伴侣蛋白FkpA的过表达、培养条件和分子设计对可溶性单链TCR的影响。升高水平的FkpA允许周质可溶性scTCR表达，推测是通过防止过早聚集和包涵体形成。周质表达能够形成二硫键，这是scTCR达到其正确折叠的先决条件。它还可以快速轻松地回收正确折叠的蛋白质，而无需耗时的下游加工。无IPTG诱导的表达进一步提高了周质表达产率，而向生长培养基中添加蔗糖几乎没有效果。获得mg水平的活性纯化材料的摇瓶产率。V α β结构域的方向是远远优于上级的V β α结构域的方向就单体产量的功能性折叠molecules.Conclusion：一般表达制度，这里提出的允许快速生产可溶性scTCR和适用于1）高产量回收足够的生物物理特性和2）高通量筛选这样的分子分子后的分子工程。
Background: 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.Results: 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 V alpha beta domain orientation was far superior to the V beta alpha domain orientation regarding monomeric yield of functionally folded molecules.Conclusion: 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.