Selection of potentially optimized trypsin variant with a more hydrophobic cluster active site via a phage display approach

Abdul Kudos, Muhammad Badri and Gissel, Sabrina and Bordusa, Frank (2015) Selection of potentially optimized trypsin variant with a more hydrophobic cluster active site via a phage display approach. In: ASIAN Congress on Biotechnology 2015, 15-19 Nov 2015, Kuala Lumpur. (Unpublished)

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Abstract

Protein optimization can generally be obtained by rational enzyme design and directed evolution. Phage display has been used as a method for directed evolution. Trypsin has been studied as a biocatalyst in peptide synthesis despite its usual function in hydrolyzing peptide bonds. The objective of this study is to identify trypsin variants with a more hydrophobic cluster around its active site, potentially yielding higher peptide synthesis products rather than its hydrolysis competitive counterpart. A trypsin variant library displayed by phages with randomization in 5 amino acids located around the active site of the trypsin variant K60E/N143H/E151H/D189K is selected based on a designed immobilization-elution-assay in regard the transamidation reaction model. A preselection stage of recombinant phages was done with MyCUT tag, binding to the immobilized anti-c-myc antibodies. 2 trypsin variants were successfully expressed, purified, characterized and sequenced. Both of them gave a higher accumulative hydrophobicity relative index. This study has shown that selection of a potentially optimized trypsin variant with a more hydrophobic cluster at active site to reduce the hydrolysis products is possible by using the phage display approach.

Item Type:	Conference or Workshop Item (Poster)
Additional Information:	6521/48397
Uncontrolled Keywords:	Phage display, Protein optimization, directed evolution, biocatalysis, protein engineering, peptide synthesis
Subjects:	T Technology > TP Chemical technology > TP248.13 Biotechnology
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button):	Kulliyyah of Pharmacy > Department of Pharmaceutical Technology
Depositing User:	Muhammad Badri Abdul Kudos
Date Deposited:	01 Jun 2017 16:12
Last Modified:	01 Jun 2017 16:12
URI:	http://irep.iium.edu.my/id/eprint/48397

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