Tjhung Katrina F, Deiss Frédérique, Tran Jessica, Chou Ying, Derda Ratmir
Department of Chemistry, Alberta Glycomics Centre, University of Alberta Edmonton, AB, Canada.
Front Microbiol. 2015 Apr 28;6:340. doi: 10.3389/fmicb.2015.00340. eCollection 2015.
In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).
在本文中，我们描述了丝状噬菌体M13的pIII蛋白上多价展示肽和蛋白质序列的方法，这些序列通常在传统的N端展示中被截短。通过对市售M13KE噬菌体克隆载体进行定点诱变，我们引入了一些位点，允许使用限制酶在pIII蛋白的N1和N2结构域之间进行高效克隆。由于噬菌体的感染性与N1和N2结构域之间连接的完整性直接相关，域内噬菌体展示（ID-PhD）允许对展示进行简单的质量控制以及所展示序列的自然变异。此外，与噬菌体繁殖的直接联系允许对序列切割进行有效监测，为蛋白酶敏感或蛋白酶抗性序列的选择和进化提供了一个方便的系统。作为这种ID-PhD系统优势的一个例子，我们展示了一个带负电荷的FLAG序列，已知当在N端展示时该序列会在翻译后从pIII上切除，以及带正电荷的序列，当在N端展示时会抑制噬菌体的产生。FLAG的ID-PhD表现出亚纳摩尔级的表观Kd，表明展示具有多价性质。与FLAG串联共表达的烟草蚀纹病毒（TEV）蛋白酶识别序列（TEVrs），使我们能够证明99.9997%的噬菌体展示了FLAG-TEVrs串联体，并且可以被TEV蛋白酶识别和切割。剩余的0.0003%由从其基因组中切除插入片段的噬菌体克隆组成。ID-PhD也适用于展示蛋白质微结构域，例如蛋白A的33个残基最小化Z结构域。我们表明，因此可以使用ID-PhD进行微结构域蛋白（亲合体、单链抗体等）的多价展示和选择。
