Generation of systems for the synthesis of recombinant proteins for use in rapid

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

Neutrophils are an essential defense force of the immune system and the most abundant type of white blood cell in mammals. Whilst they play an essential role in protecting the body from attack by foreign agents (e.g. microbes) they can also destroy the tissue that they protect. One method by which they accomplish this is via the release of proteases that literally destroy the tissue by 'digesting' it. For example, proteases that are released by neutrophils are matrix metalloproteinases (MMPs)-8 and -9. MMP-8 and -9 are collagenase cleaving enzymes. Work at Mologic has shown that the presence and amounts of these enzymes can be used to confirm the presence and severity of neutrophil infiltration. The proteoyitc activity can therefore be used as a biomarker of infection and inflammation, particularly in non-healing wounds. Here we intend to investigate development of systems for the rapid development of recombinant materials for use in diagnostic technologies. The development of diagnostic technologies for point-of-care use requires pure recombinant proteins rapidly at lab scale at an affordable cost. As the majority of these proteins are human proteins they often require post-translational modifications such that they are as close to human-like as possible. This means that expression in bacterial systems is often not possible. In this project the student will develop systems for the rapid evaluation of bacterial (E.coli) and mammalian expression of target recombinant proteins, specifically MMPs, and the subsequent purification of these and application into diagnostics of neutrophil infiltration. At Kent the student will initially develop bacterial and mammalian expression vectors, with mammalian expression vectors that allow for the expression of up to four recombinant genes simultaneously from the same plasmid/vector transiently and stably with appropriate tags for purification (milestone 1). These plasmids will then be used to not only clone and express the recombinant target genes of interest, but also genes to aid in the folding and secretion of the target protein from mammalian cells to improve the quality of the proteins (milestone 2). The influence of co-expressing translation initiation and elongation factors, ER chaperones and vesicle SNAREs along with the target proteins will be evaluated in terms of the amount and quality of recombinant protein expressed from CHO cells. This will be compared to expression levels and the quality of the product in E.coli (milestone 3). The final stage of the project will be undertaken at Mologic and involve the evaluation of the recombinant MMPs in diagnostic assays (milestone 4). The outcomes of this project will be the development of systems for the rapid expression of quality human-like proteins for use in diagnostics and an understanding of how engineering of translation, ER processing and secretion simultaneously influences protein expression and quality.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M017060/1 01/10/2015 30/09/2019
1666804 Studentship BB/M017060/1 19/09/2015 30/09/2019 Andrew David Martin
 
Description We have achieved expression and purification of soluble Elastase, a secreted protease and known virulence factor of the gram negative bacterium Pseudomonas aeruginosa.

We have successfully generated sheep and rabbit polyclonal antibodies to Elastase. These antibodies have been incorporated into two diagnostic assays; a lateral flow assay and an enzyme-linked immunosorbent assay, able to detect Elastase at concentrations of < 100 pg/mL with no observed cross-reactivity with other pseudomonas proteins or proteins secreted by other gram negative bacteria. This assay has been tested on clinical isolates obtained from cystic fibrosis patients in collaboration with Imperial College London.

We have attempted to generate high affinity monoclonal Fab and scFv fragments specific to elastase via antibody phage display. However, despite generating large, diverse immune libraries, we have currently been unable to successfully select soluble monoclonal fragments.
Exploitation Route The anti-elastase diagnostic assay that we have been developing, once finished, would ideally be used in the clinic to detect early stage Pseudomonas aeruginosa infections in Cystic Fibrosis patients. However, more work must be performed using clinical samples in order to determine if it is a viable assay.

Further development of the assay could be performed through the generation of monoclonal antibody fragments via antibody phage display, as well as generation of antibodies to other pseudomonas virulence factors in order to produce a multiplex assay, if the detection of elastase alone is not considered a reliable diagnostic test.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology