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Bacteriophage display of peptides and its use in determining proteolytic substrate specificity
C. Carrie Liu1, Joti Nahal1, Kim Melton1, Navneet Sharma2
1Faculty of Medicine, University of Calgary, Calgary (AB) Canada.
2Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary (AB) Canada.

Article ID: 100001M11CL2015
doi:10.5348/M11-2015-1-RP-1

Address correspondence to:
Navneet Sharma
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary
Calgary (AB), T2N 1N4
Canada
Phone: 403-210-7079

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How to cite this article
Liu CC, Nahal J, Melton K, Sharma N. Bacteriophage display of peptides and its use in determining proteolytic substrate specificity. Edorium J Mol Biol 2015;1:1–5.


Abstract
Introduction: Phage display has been employed for many applications including generating highly specific antibodies, determining protein-protein interactions and substrate specificity determination. We have recently used peptide display on T7 phage to successfully determine the substrate specificity of serine proteinases (Sharma et al., 2008, 2011 and 2013). The random phage library we made displays a hexameric peptide on the surface using the library construction described by Karlson et al. (2002) with minor modifications. Each phage particle is able to display no more than one peptide on its surface. Therefore, it is a unique library with each recombinant phage particle displaying a random peptidic substrate on the surface. The library represents all the possible combinations of random hexamers (6.7×107) as described by Deperthese (2002).
Protocol: In this article, we demonstrate the process of building the phage library with random peptides displayed on the surface and using it for determining the substrate specificity of a proteolytic enzyme by repeated rounds of biopanning. The phage library being built is a random one with each phage particle displaying a random peptide on its surface. The library is sequenced after constructing and the frequency of display is noted down. The ratio of displayed amino acid (expected vs. displayed) is determined and it is well within the range (i.e. 0.5–2.0) as shown by Cwirla et al. (1990), confirming the randomness of the library.
Conclusion: In order to check the validity of this library, we have tried to find peptidic substrates for a known serine proteinase i.e. trypsin in this paper. All the substrates found for tryspin are known, thus confirming the validity of this library. The aim of this paper is to show an innovative technique for the display of substrate peptides on the surface of bacteriophage and their random selection based on substrate specificity of a protease. It can be applied to any protease for determining its substrate specificity as we have done.

Keywords: Phage display, Substrate specificity, T7 Phage, Serine proteinase, Oligonucleotide, Phage plaques, Polymerase chain reaction (PCR)

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Author Contributions:
C. Carrie Liu – Substantial contributions to conception and design, Acquisition of data, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Joti Nahal – Analysis and interpretation of data, Revising it critically for important intellectual content, Final approval of the version to be published
Kim Melton – Analysis and interpretation of data, Revising it critically for important intellectual content, Final approval of the version to be published
Navneet Sharma – Analysis and interpretation of data, Revising it critically for important intellectual content, Final approval of the version to be published
Guarantor of submission
The corresponding author is the guarantor of submission.
Source of support
None
Conflict of interest
Authors declare no conflict of interest.
Copyright
© 2015 C. Carrie Liu et al. This article is distributed under the terms of Creative Commons Attribution License which permits unrestricted use, distribution and reproduction in any medium provided the original author(s) and original publisher are properly credited. Please see the copyright policy on the journal website for more information.



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