PeriTune – A Clonal Optimisation Platform
Blog Feb 27, 2015
A joint project between Cobra Biologics and The University of Manchester has recently been awarded £217K funding, as part of a UK initiative to develop industrial biotechnology.
To learn more about the project and what it hopes to achieve, we spoke to Tony Hitchcock, Technical Director at Cobra Biologics.
AM: Cobra Biologics, alongside The University of Manchester, has been awarded £217K funding from the Innovate UK Industrial Biotechnology Catalyst competition. Can you tell us more about this initiative?
TH: The initiative is a £20m fund to support the development of Industrial Biotechnology within the UK and is supported by the Biotechnology and Biological Sciences Research Council (BBSRC), the Technology Strategy Board (now Innovate UK) and the Engineering and Physical Sciences Research Council (EPSRC). There are five types of awards available – early stage: translation; early stage: feasibility studies; industrial research; late stage: pre-experimental feasibility studies; and late stage: experimental development. The programme awarded to The University of Manchester (UoM) and Cobra Biologics (Cobra) relates to the initial feasibility programme.
AM: The funding has been awarded for the PeriTune project. What are some of the aims of this project?
TH: The overall aims of the project are to develop a low cost high throughput screening for molecular constructs and fermentation conditions for the production of novel recombinant proteins within the cellular periplasm in bacterial systems. Whilst there are existing high throughput screen approaches available to identify expression candidates, they are based on complex expensive robotic systems, and require multiple rounds of screening. The aim of this project is to combine the two capabilities to give a powerful low cost screen approach with increased success rates than are normally achieved with these approaches.
From a work programme perspective, this will encompass the development of high throughput molecular biology based screening systems to identify optimal amino acid leader sequences for model therapeutic protein products to cellular periplasm. In parallel Cobra will transfer in house the RiboTite technology from the UoM and develop bacterial hosts strains for therapeutic protein products by introducing the RiboTite expression system into established productions strains. The outputs from the high throughput screen will then be assessed in the newly generated production strains at Cobra.
AM: The project will utilise RiboTite technology, developed at The University of Manchester. Can you explain how this technology works, and the role it will play in the project?
TH: The RiboTite expression system is composed of an engineered E. coli strain and series of expression plasmids. The unique feature is that it uses mRNA devices, or orthogonal riboswitches, that operate at the level of translation initiation (Dixon et al PNAS 2010). This feature allows tight control of basal expression in the absence of induction and tuneable control of expression across a broad dynamic range.
In this current PeriTune project we will be applying the tuneable capabilities of RiboTite to match the host’s secretion capacity. In applying the technology to secretion optimisation as part of the project we will be further fine tuning parts of the expression system.
AM: Why is it advantageous to control expression of a target protein to occur only in the periplasm, rather than in the cytoplasm?
TH: Traditional approaches for protein production in bacterial systems frequently result in the formation of inclusion bodies (aggregates highly-enriched with the protein of interest) in the cytoplasm of the host cells. Whilst these inclusion bodies can be further processed (and are in a lot of cases) this requires additional processing steps (increasing time and cost). Furthermore, a lot of the novel and new types of protein therapeutics that we are being asked to manufacture require the formation of internal disulphide bonds to impart structural stability and correct function on the molecule. These types of bonds do not form efficiently (or at all in some case) within the cytoplasm of E.coli, but do in the periplasm the bacteria. Therefore, we are looking to develop a system that allows for fine control of expression of a target protein coupled to periplasm targeting, in order to overcome the burst of expression and also to allow for the correct formation of disulphide bonds. An additional benefit is that the protein content of the periplasm is less enriched in contaminating proteins then the cytoplasm which can also help with the purification steps during the manufacture.
AM: How will the collaboration benefit Cobra Biologics?
TH: Much of Cobra business is based around the development and provision of manufacturing solutions to our customers often seeking to bring new and innovative protein products to the clinic and an increasing proportion of these require microbial based production routes. This approach and technology will offer significant improvement to Cobra’s technical offering we can offer customers, which in turn will hopefully translate to increased business success in this field. Since its establishment over 20 years ago, Cobra has always sought to develop long term partnerships with leading academic groups within the UK to enhance its capabilities and has been involved in a number of successful collaborations in the past, and we hope that this will project will lead to a long term relationship with the UoM to further enhance and develop this exciting technology.