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Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

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Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display

Katrina F Tjhung¹, Frédérique Deiss¹, Jessica Tran¹

¹Department of Chemistry, Alberta Glycomics Centre, University of Alberta Edmonton, AB, Canada.

Frontiers in Microbiology

|May 15, 2015

Abstract:

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.).

Insights

This study introduces intra-domain phage display (ID-PhD) for improved peptide and protein display on M13 bacteriophage. ID-PhD enables robust multivalent display and selection of sequences, overcoming limitations of traditional methods.

Area of Science:

Molecular Biology
Biotechnology
Protein Engineering

Background:

Traditional N-terminal display on filamentous bacteriophage M13 protein pIII faces limitations with certain peptide and protein sequences.
Post-translational excision and production suppression hinder the display of specific charged sequences.
The integrity of the N1-N2 domain connection in pIII is crucial for phage infectivity and display quality.

Purpose of the Study:

To develop a novel phage display system, intra-domain phage display (ID-PhD), for multivalent display of challenging peptide and protein sequences.
To enable efficient quality control and monitoring of sequence cleavage for selection and evolution.
To demonstrate the utility of ID-PhD for displaying sequences typically censored from N-terminal display and for selecting mini-domain proteins.

Main Methods:

Site-directed mutagenesis of the M13KE phage cloning vector to introduce cloning sites between pIII protein domains N1 and N2.
Utilizing the linkage between infectivity and the N1-N2 domain for quality control and monitoring sequence cleavage.
Co-expression of FLAG and TEV-protease recognition sequence (TEVrs) for assessing display efficiency and protease susceptibility.

Main Results:

ID-PhD successfully enabled multivalent display of negatively charged FLAG sequences with sub-nanomolar apparent Kd.
Demonstrated efficient monitoring of protease cleavage, with 99.9997% of phage displaying the FLAG-TEVrs tandem.
Showcased the display of protein mini-domains, such as the minimized Z-domain of protein A, indicating suitability for Affibodies and scFv.

Conclusions:

Intra-domain phage display (ID-PhD) offers a robust platform for multivalent display of peptide and protein sequences.
ID-PhD overcomes limitations of traditional phage display, allowing for the display of otherwise censored sequences and efficient selection.
This method is versatile for displaying and selecting various protein mini-domains, expanding applications in protein engineering and drug discovery.

Read full analysis

Keywords:

M13 bacteriophage excised peptide in vitro selection phage display protein engineering

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