Preclinical Research and Development, XOMA Corp., Berkeley, CA 94710, USA.
J Immunol Methods. 2013 Aug 30;394(1-2):10-21. doi: 10.1016/j.jim.2013.04.010. Epub 2013 Apr 23.
Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.
不正确的蛋白质折叠或聚集常常导致抗体片段在大肠杆菌周质中分泌时表达量低且功能活性差。表达问题也会影响从噬菌体文库中选择抗体候选物，因为噬菌体上展示的抗体片段也分泌到大肠杆菌周质中。为了提高正确折叠的抗体片段分泌到周质中，我们开发了一种新方法，涉及与肽基脯氨酰顺反异构酶 FkpA 共表达，该酶缺乏其信号序列（cytFkpA），因此在大肠杆菌细胞质中表达。细胞质中 cytFkpA 的表达提高了功能性 Fab 片段分泌到周质中的效率，甚至超过了与位于周质中的天然 FkpA 共表达 Fab 片段的效果。此外，在存在 cytFkpA 的情况下对大的 Fab 和 scFv 原始噬菌体文库进行淘选和后续筛选，显著增加了所选独特克隆的数量及其功能表达水平和多样性。
