Overview

Biotechnology

The Biotechnology group is primarily responsible for generating the pipeline of clones targeted by the SGC in Oxford and determining using high-throughput (HTP) screening methods which proteins are expressed in a soluble and stable form suitable for structural and functional studies. We have developed and optimised protocols for high-throughput cloning, test expression/purification and mass spectrometry analyses, large scale expression and protein production and validation. We screen a wide range of proteins (intracellular, extracellular and integral membrane proteins) using E. coli, baculovirus/insect cell and mammalian (BacMam) expression systems. The platform established by the Biotechnology team, has enabled the site at Oxford to generate more than 550 novel human protein structures and 5 integral membrane protein structures. The group collaborates and interacts closely with the other SGC teams, to develop methods for increasing protein expression, parallel processing and driving output. 

Ligation Independent Cloning (LIC)

We have developed a 96-well format for PCR and cloning which does not rely on robotics. We employ LIC cloning as our preferred method for generating SGC constructs which gives us the flexibility to clone any gene insert into our wide selection of LIC-adapted vectors. Our HTP methods can be performed in any laboratory using multichannels pipettes.

High-Throughput (HTP) Screening

  

We have established HTP screening platforms for bacterial, baculovirus and mammalian expression systems. Using these methods, we can process 96 constructs in parallel to identify soluble proteins for further scale up work and analysis. We can also apply these HTP methods to screen integral membrane proteins (IMPs). By applying a two-stage process, firstly purifying with n-dodecyl-β-maltoside (DDM), followed by a second screen against a panel of detergents and cholesterol hemisuccinate (CHS), we can identify stable conditions for proteins suitable for further scale up and characterisation.

Baculovirus Expression

Our established baculovirus facility and HTP screening platform have allowed us to overcome problems with expressing more challenging proteins such as kinases and IMPs. The baculovirus expression team produce up to 600 L of baculovirus-infected insect cells for the Biology groups each month.

Parallel Protein Production

We have developed platforms for large scale expression and purification of soluble proteins from E. coli and baculovirus/insect cells. Using ÄKTA-Xpress systems, we have the capability to purify 12-16 proteins in parallel overnight.

Mass Spectrometry

The mass spectrometry facility provides protein characterisation for the SGC’s high-throughput crystallography, chemical probe discovery and epigenetics programs. We develop new methods and analytical capabilities in response to the specific needs of SGC scientists. Current equipment includes Agilent 6530 QTOF, Agilent MSD-TOF, Agilent 6530 Rapidfire QTOF and Bruker HCT ion trap mass spectrometers. We have specific expertise in native mass spectrometry, characterisation of intact soluble proteins and integral membrane proteins due to the SGC’s unique collection of expressed human proteins. Our current research interests include:

  • High-throughput characterisation of proteins from small scale test purifications
  • Characterisation of protein folding, stability, protein complex formation and ligand binding
  • Intact and fragmentation analysis of protein post-translational modifications
  • Integral membrane proteins and their associated ligands, lipids and detergents
  • Measuring enzymatic activity via high throughput peptide quantitation

We collaborate with other research groups both at Oxford University and globally

Methods Development

The Biotechnology group is also interested in testing and developing new methods for improving expression, purification and crystallisation of all SGC proteins. We currently have projects ongoing for the following :

  • Expression of complexes in E. coli and baculovirus/insect cells
  • Expression and purification using BacMam
  • Improving expression levels of IMPs
  • Miniaturisation to increase throughput & reduce screening time (using analytical gel filtration (GF) to generate protein for crystal trials)
  • Crystal epitope mutagenesis
  • Reducing costs for the SGC (comparative studies using alternative cheaper sources of reagents/media, etc.)

Group Members

Rod Chalk

Rod Chalk gained his PhD in 1992 at The Liverpool School of Tropical Medicine, working on the innate immune peptides from the mosquito Aedes aegypti.  He began using mass spectrometry for bioactive peptide natural product drug discovery at Queens University of Belfast in 1996, subsequently working on SNP genotyping and cryodetector mass spectrometry. More recently, Rod has worked in proteomics and biopharmaceuticals using a variety of techniques and instruments. Rod joined the SGC in 2008. His current interests include high-throughput protein characterisation, membrane proteins, quantitative mass spectrometry, and the application of native mass spectrometry to structural biology and drug discovery.

Leela Shrestha

Leela Shrestha received her M.Sc. in Medical Microbiology from Tribhuvan University, Nepal. She then worked as Microbiologist and Teaching assistant in People’s Dental college and Hospital, Nepal. Leela joined the SGC in 2006 to work on expression, purification and crystallisation of human proteins. In 2008 she became involved in screening, expression and purification of human membrane proteins expressed in E coli and insect cells and more recently has been focussed on high-throughput screening of both integral membrane proteins and soluble proteins for the Biotech group.

Shubhashish Mukhopadhyay

Shubhash joined the SGC in March 2011, with a primary focus on baculovirus expression of membrane proteins, HTP detergent screening and assisting with method development projects for the team. His inputs in the eukaryotic cell pipeline for membrane proteins along with maintenance of the cell culture facilities, has resulted in the deposition of membrane protein structures over the last few years. Previously, he worked at Reckitt Benckiser Plc., as a research analyst, working on product stability analysis, process validation and development projects in a cGMP environment till 2008 and has also worked at Oxford Expression Technologies Ltd. as a quality control assistant working on production and quality testing of recombinant bacmid DNA. Since 2017, Shubhash works as a Research assistant- cell culture & facilities coordinator, and has an oversight on the insect and membrane protein expression pipeline. He is working closely with the membrane protein groups on troubleshooting and helping the mammalian expression pipeline. He is grateful to have an amazing array of talented people around him and loves to train others and learn himself. He plans to develop his career into the clinical research environment, working in academia/industry.

Aleksandra Szykowska

Aleksandra Szykowska is a D.Phil student working on a project funded by pharmaceutical company Eisai. She is investigating the role of microglia immune receptors in the context of Alzheimer’s disease in SGC and Oxford drug discovery institute (ODDI). Earlier she completed her M.Sc. in molecular biology at Warsaw University. Her early experience involved RNA processing machinery complexes, mammalian microRNAs and yeast genetics. During her M.Sc. Aleksandra was expressing and purifying proteins and undertaking biophysical and functional characterisation assays of yeast THO/TREX complex. She joined SGC in 2012 as a protein purification research assistant.

Dong Wang

Dong obtained a Master’s degree in International Nutrition and Food Processing in 2006 and spent the following year working as a research assistant in Oxford Brookes University. Dong commenced working for the University of Oxford in 2008 on mammalian cell culture and the quality control of Adenovirus. Dong joined the SGC in 2013 as a research technician. She is currently responsible for high-throughput cloning, test expression and purification of soluble proteins and membrane proteins. She also works on large-scale Baculovirus expression and is involved in high-throughput screening of mammalian cells.

Gavin McKinley

Gavin was a vehicle mechanic until redundancy presented an opportunity for a career change. In 2001, he joined the Clinical BioManufacturing Facility as a production assistant. This is the University of Oxford’s GMP facility for the pilot scale production of therapeutic drugs. His role included mammalian cell culture, media preparation, autoclave operation and environmental monitoring of the clean rooms. In 2008 he moved abroad for a couple years before returning and joining the SGC in 2014 as a lab support technician. In 2015, he joined the Biotech group where he assists with the baculovirus protein expression pipeline, clone distribution and occasional lab support.

Álvaro Viñals Guitart

Álvaro graduated from his M.Res. in Bioengineering at Imperial College in 2016, where he worked with acrylated agarose scaffolds to enhance cartilage regeneration via modification with TGF-β. Prior to this he had spent a year working as a Research Assistant at the Center of Biomaterials and Tissue Engineering in the Polytechnic University of Valencia. His responsibilities comprised the synthesis and characterisation of alginate-chitosan and magnetic poly-lactic acid microspheres for drug delivery and cartilage repair. He joined the SGC in a joint position with the Kennedy Institute of Rheumatology where he works in the structure of a protein complex involved in tissue regeneration as part of his D.Phil project.

Alejandra Fernandez-Cid

Ale obtained her PhD in Molecular Biology and Biotechnology at the University of Oviedo, Spain, in 2011. Her thesis involved the characterisation of several transcriptional repressors and their role in the crosstalk between glucose metabolism and mitochondrial morphology. By the end of that year she joined the MRC (London Institute of Medical Sciences) as a postdoctoral researcher. During this first postdoc she worked in the eukaryotic DNA replication field, focusing on the in vitro reconstitution of the pre-replication complex and its subsequent cryo-EM analysis. In 2012, she moved to Imperial College where she continued working with eukaryotic DNA binding proteins. Her aim was to help unravel the mysteries of chromosome condensation, particularly the role of histone PTMs and condensin. Ale joined the SGC in 2017 where she manages the HTP screening pipeline under Nicola. Her main interest is to keep it running in a smooth and efficient way and improve it when possible. She loves being surrounded by a great team of people J

Edward Bower

Edward graduated from The University of Edinburgh in 2010, with a BSc in Biological Sciences (Biochemistry Hons). Continuing the work from his Honours project, he then completed a Masters by Research in enzymes from the sphingolipid biosynthetic pathway. Remaining at Edinburgh, Edward joined the group of Dr David Dryden in 2012. Here, he carried out his PhD on the evolution of Restriction-Modification systems, in collaboration with Dr. Richard Morgan (New England Biolabs). Edward joined the SGC in 2018, to take a research assistant position in the Biotechnology group. His main responsibilities are cloning, test expression and protein purification.

Shayla Venkaya

Shayla Venkaya completed her BSc degree at Oxford Brooke’s University and then went on to complete a Postgraduate Degree in Immunology in 2017 at King’s College University. Shayla joined the SGC shortly after as a Research Technician, currently working on large scale baculovirus expression in insect cells. She is also involved in high-throughput cloning, test expressions and purifications of membrane proteins.

Patrizia Abrusci

Patrizia’s research training started in Italy with a Master degree in Biology and then was consolidated with a PhD in Biotechnology and Biomolecular Science, at the University of Pavia, where she investigated the relationship between structure and function of defective enzymes in the nucleotide homeostasis pathway of red blood cells. Subsequently, she enriched her academic path with a postdoc at the MFPL, University of Vienna, joining Prof. Kristina Dijnovic’s team, focused on the structural biology of Z-disk proteins. At MFPL she became skilled in protein crystal handling, soaking, optimizations and X-ray diffraction and data collection. When she moved to Oxford, working for several years as a Research Associate at the Dunn School of Pathology, she acquired expertise in protein expression and purification of membrane protein complexes in a quite broad array of forms (from detergent micelles to liposomes and detergent free polymer nanoparticles), suitable for crystallography and Cryo-EM. Patrizia joined the SGC in 2018 where she currently works on the characterization of a membrane protein complex involved in tissue regeneration.

Tina Bohstedt

Tina joined the SGC in November 2018, working jointly between the SGC and the Kennedy Institute of Rheumatology. She is involved in cloning, expression and purification of membrane proteins.

Niyalish Fatimah

Niyalish was selected by the Nuffield foundation to partake in a 1-month research project. This was her first exposure to a research laboratory. Subsequently, during her undergraduate degree she carried out molecular techniques such as reverse transcription, site directed mutagenesis, mammalian cell transfections and xenopus oocyte injections to investigate novel patient mutations. Niyalish’s Master’s research involved investigating a novel spliceosomal mutation which causes a rare developmental syndrome. She trained in advanced techniques such as immunoprecipitation and minigene assays with patient derived fibroblasts. Shortly before Niyalish obtained her Master of Research degree in Translational Medicine, she was fortunate enough to join Biotech in September 2018. Her main role is to express soluble proteins at a large scale in an insect cell expression system using the baculovirus system. Niyalish also has the responsibility of distributing SGC clones nationally and internationally. She loves being part of the best team in the SGC.

Tiago Moreira

Tiago obtained his MSc in Chemistry at the University of Porto in 2014 and has done an “Erasmus” internship at the Section of Forensic Toxicology, University of Copenhagen. In 2015, he was a trainee at the European Medicines Agency where he contributed to the regulatory work of medicines certification at the Department of Compliance and Inspections. From 2016 to 2018 he worked in the pharmaceutical industry as a medical device test technician at the Department of Research and Development at Owen Mumford and as a trainee analyst at Abbott. Tiago joined the SGC in 2018 as a mass spectrometry technician and is currently involved in high-throughput mass spectrometry analysis of soluble and membrane proteins.

Rama McCrorie

Rama completed her PhD in cell and molecular biology from the University of Essex in 2014 where she worked on developing single molecule imaging assays. She then moved to the Imperial College London for her postdoctoral post in malaria research. For her postdoctoral position, she worked on developing expression and purification methods of malaria parasite’s proteins. She started working at UCB Celltech in 2018 as an assay development scientist before moving to Oxford drug discovery institute (ODDI) in 2019 for her next postdoctoral position in protein purification. ODDI is closely associated with structural genomic consortium (SGC) where she works closely with the Biotechnology unit to produce proteins for assays and screening.

Leslye Roca Burgos

Leslye obtained her PhD in Natural Sciences from the Structural Biology Group at the Christian-Albrechts-University of Kiel in Germany in July 2018. Her PhD research focused on protein expression in bacterial and insect cells, purification, crystallization and biophysical characterization of ATP-independent Luciferase from Gaussia princeps. During this period, she gained experience working with enzymatic activity assays to characterize the function and regulation of the target protein. Prior to her PhD she gained research experience at the EMBL-Hamburg in Germany working with recombinant protein production for structural studies from Mycobacterium tuberculosis targets, and at the Liver Research Centre in Italy working with multidrug resistance associated protein expressed in eukaryotic cells. She joined the SGC in 2019 as a Postdoctoral Scientist to support protein production for internal projects and external collaborations.

Adam Mabrouk

Adam joined the Biotechnology group in 2019. His primary role is to generate expression constructs using high-throughput cloning and perform protein expression/purification in E. coli, insect and mammalian cells. He received a First Class Honours in Biochemistry from the University of Surrey in 2018 and previously spent a Professional Training Year (PTY) at Oxford University working at the Sleep and Circadian Neuroscience institute (SCNi). This research involved looking at melanopsin (OPN4) expressing photosensitive retinal ganglion cells (pRGCs) and how they mediate physiological responses to light in mammals. After a brief experience in industry, he returned to Oxford University to pursue his passion of working in an academic environment. Adam is a former Royal Marine Commando. He served in the Royal Marines from 2002 – 2011. He left the Military to embark on a career in science, which he believes to be a privilege.

Alexander Stephens

Alex obtained his BSc. Biochemistry degree in 2012 from the University of Southampton. Following graduation he worked for 5 years within the biotechnology sector. Firstly, he worked until 2015 as Production Technician for a biotech company based near Oxford, and was involved in producing antigens for the In vitro diagnostic (IVD) and Vaccine development industry. Subsequently, from 2015 he worked as a Scientist in the cell line development group for a biotech company in Cambridge, there his role was to generate stable-transfected mammalian cell lines for the production of monoclonal antibodies. In 2019, Alex Joined the Oncology group as a Research Assistant, prior to commencing his DPhil, for Shisong Jiang in Len Seymour’s group. He is working closely with the SGC (co-supervised by Nicola Burgess-Brown) in the production and purification of recombinant overlapping peptides (ROPs).

Group Alumni

Pravin Mahajan, Hannah Brazier, Oktawia Borkowska, Claire Strain-Damerell, Katherine Ellis, Katarzyna Kupinska, Matthias Ehebauer, Maciej Kliszczak

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