Sortase– Mediated Transpeptidation: The Application Of Sortase A As A Tool For Site- Specific Modification Of Recombinant Antibodies
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Date
2016-03
Authors
Ismail, Nur Faezee
Journal Title
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Publisher
Universiti Sains Malaysia
Abstract
Point-of-care (POC) diagnostics for resource deprived regions remains a major challenge to combat infectious diseases worldwide. Development of new methods to aid the development process of new POC diagnostics is essential to improve the healthcare scenario worldwide. The major format of POC assay is the detection of antibody-antigen interaction. To achieve this, an assay is required to be capable of reporting the interaction between the antibody and antigen. In most common assays, these detection are achieved by conjugating antibodies to specific reporter molecules such as enzymes or proteins. The work describes the application of sortase mediated transpeptidation as a means to conjugate recombinant antibodies to a pair of reporter proteins. Sortase enzymes are unique in a way that they recognized a specific sequence motif for reaction allowing a site-specific conjugation of proteins. Due to this specificity, they are very beneficial to be used for bioconjugation mainly in developing diagnostic assay platforms. SrtA (0.546kb) was isolated from Staphylococcus aureus and cloned inside pRSET system under a tight control of T7 promoter. A set of motif were also fused, cloned and expressed to select the most efficient motif at an optimized condition. A proof-of-concept experiment was conducted by using the anti-ubiquitin scFv(0.72kb) as a donor motif (LPXTG motif) protein. Meanwhile, enhanced green fluorescent protein (eGFP)(0.714kb) and invertase(1.236kb) were chosen as the receiver motif (glycine motif) protein functioning as the reporter system. A double tag purification strategy was developed to improve purification yield of the conjugated product. Development of two different platforms utilizing two different reporter proteins was used. Initial investigation was carried out using eGFP for application on a fluorescent immunoassay platform. After optimizing the ideal conditions, a second conjugation process was developed for invertase enzyme. The conjugation to invertase allowed for rapid development of antibody-antigen assays detectable using a personal glucose meter (PGM). The function of conjugated invertase was to generate a readout on the PGM in the presence of glucose. The sortase mediated conjugation process allows for an easy and rapid development of recombinant antibody derived POC assays on the PGM platform. This would provide an interesting alternative for the development of antibody-antigen assays utilizing a PGM for POC applications.
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Keywords
Antibodies