Cambridge Healthtech Institute’s Ann Nguyen recently interviewed Nicola Burgess-Brown of the University of Oxford. Dr. Burgess-Brown shares her keynote presentation on “Expressing Human Intracellular and Integral Membrane Proteins for Structural and Functional Studies” at the Recombinant Protein Expression conference taking place January 19-20, 2016, as part of the 15th Annual PepTalk in San Diego, CA.

Q1: What’s it like working at Oxford’s Structural Genomics Consortium? What kind of environment and resources do you have there for developing methods that increase protein expression?

The SGC is a great place to work. It promotes research advancement through our open access policy, providing knowledge and tools freely available and not having to worry about IP. I started my research career in industry so moving to the SGC was a gentle transition into academia largely because our high-throughput (HTP) approaches and pipeline processes for making proteins are very similar to those used by large pharma and biotech. The SGC is a unique mix of industry-standard combined with the academic freedom to do research. I have worked at the SGC for 11 years and during that time the organization has grown and adapted from solving the 3-dimensional structures of human proteins to developing chemical tools (probes) and affinity (antibody) reagents for epigenetics targets. These reagents, provided freely to the research community enable better understanding of protein function and ultimately better drug design.

More recently, through the acquisition of our new grants, we are now expanding from protein structures and chemical probes to developing assays, screening compounds in human tissue platforms and working with patient groups with the ultimate aim of taking a starting compound all the way to clinical trials. All of this, with our open access policy leading the way.

The collaborative nature of the SGC staff and our close interaction with our pharma funders and academic collaborators ensure that all the partners benefit from the knowledge gained. We openly discuss/share methodologies, both internally and externally to avoid duplication of effort. In my opinion, this is how research should be done. Internally, we work in parallel using systematic procedures. My group, for example, performs test expression and purification on all SGC targets and we are responsible for the major insect cell scale-up expressions for the Oxford site. The Toronto site works in a similar way. This enables smooth-running facilities and allows each group to focus on what they do best. The knowledge and experience of the staff is a key factor in our success and this knowledge gets passed on to new recruits ensuring that we all work optimally.

To increase protein expression, we always take the approach (as many labs do) of making multiple constructs of each protein target and testing both prokaryotic and eukaryotic expression systems (namely E. coli and Baculovirus). To account for very challenging proteins which may require specific post-translational modifications, we have developed a mammalian expression system (BacMam), both for HTP screening and large scale protein expression. We will continue to develop new methods such as this to enable production of as many human proteins as possible in the future.

Q2: Why have you focused on the production of human integral membrane proteins (IMPs) for structural and functional studies?

We have a large target list that comprises both soluble (intracellular) proteins and IMPs, so we have an interest in working on many different target areas (with more recent focus on targets involved in neurodegeneration, cancer, metabolism and inflammation). We started working on IMPs back in 2009. One of the main hurdles for determining the structures of IMPs is generating sufficient quantities of stable protein. There was a need to develop methods and tools to deal with these proteins, particularly as IMPs make up a significant proportion of drug targets. Less than 10 human IMP structures were available at that time but in the past 5 years, through the efforts of several labs, and the SGC’s cumulative experience with varied high-throughput methods for expression and purification, that number has increased to about 50 worldwide, with the SGC in Oxford contributing 10% of that output. Producing IMPs will continue to be a challenge but we understand their importance so we will continue to develop and optimize methods in the hope that can one day we can advance those numbers into the hundreds or even thousands.

Q3: What alternative protein expression systems show the most promise, and what kind of strategic and technological advances have you seen in recent years?

Mammalian expression seems to be most popular at the moment. Several options exist, with transient expression or production of stable cell lines often presented at conferences. We decided to use BacMam for our facility which allows us to produce virus in insect cells that will transduce and express proteins in mammalian cells, similar to the process we currently use for our Baculovirus expression system. The process transition was minimal and the results have so far been very positive. Expression of protein complexes is also on the increase as many proteins have been found to be associated with a partner or several partners and require co-expression for production. We are using both co-expression in E. coli and Baculovirus to produce protein complexes. The Multibac expression system developed by Imre Berger at EMBL has enabled the production of large protein complexes for structural biology and we have acquired and adapted this system in our lab. The interesting statistic, however, is that the majority of structures deposited by the SGC are still arising from E. coli expression. Protein engineering (mutagenesis) to improve expression, solubility and crystallization is often employed and we are now introducing this as a pipeline process to enable us to continue to work in bacteria. Our SGC site in Stockholm is generating antibodies which can be used in co-crystallization experiments to aid protein stability and promote crystallization. This technique (as well as the use of ligands) has been used successfully in structural biology for several proteins over the past couple of years. Cell-free expression has always appealed to me; to produce protein without the need for large cell cultures and lysis. In small scale, as a HTP screening tool, it seems to work very well, but the cost of scaling up may limit its application.

To learn more about Dr. Burgess-Brown’s presentation at the 18th Annual Recombinant Protein Expression conference, visit CHI-PepTalk.com/Protein-Expression-Production