Biotechnology

Group Site:

Group Leader:

Nicola Burgess-Brown

Group Info

Research Areas

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 and purification, large scale expression and protein production and validation. We screen a wide range of proteins (intracellular, extracellular and integral membrane proteins) using E. coli and baculovirus expression systems and are currently interested in piloting mammalian expression and new technologies such as bacmam. The platform established by the Biotechnology team, has enabled the site at Oxford to generate more than 360 novel human protein structures and 1 integral membrane protein structure. The group collaborates and interacts closely with the other SGC teams, to develop methods for increasing protein expression, parallel processing and driving output. We respond to the needs of the organisation to provide a smooth running and successful SGC pipeline.

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 both bacterial and baculovirus 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-ß-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 250 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. We are currently equipped with Agilent MSD-TOF and Bruker HCT ion trap mass spectrometers. We have specific expertise in 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 analysis of proteins from small scale test purifications

· Native MS characterisation of protein-protein and protein-ligand binding

· MSMS analysis of protein cross-linking

· Intact and fragmentation analysis of protein post-translational modifications

· Integral membrane proteins and their associated ligands, lipids and detergents

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 and purification of all SGC proteins.

We currently have projects ongoing for the following:

· Expression of complexes in baculovirus/insect cells

· Piloting expression in mammalian cells and bacmam technology

· Improving expression levels of IMPs

· Increasing throughput & value: using 96-well elutions from test purifications for characterisation such as intact MS, tag cleavage, etc.

· Miniaturisation to increase throughput & reduce screening time (using analytical gel filtration (GF) to generate protein for crystal trials)

· Reducing costs for the SGC (comparative studies using alternative cheaper sources of reagents/media, etc.)

Group Members

Nicola Burgess-Brown

Nicola Burgess-Brown received her Ph.D. in Molecular Microbiology (‘’Quorum Sensing and Regulation of Virulence Gene Expression in Porphyromonas gingivalis’’) at the University of Nottingham in 2001. Nicola then moved to industry to work on high-throughput cloning and validation of therapeutic cancer antigens for Oxford Glycosciences and subsequently Celltech R&D Ltd. In July 2004, she joined the Oxford node of the Structural Genomics Consortium as a Team Leader and has been responsible for optimising the high-throughput screening processes from cloning to expression and purification of human proteins. Since June 2009, Nicola has developed a similar pipeline for production of human integral membrane proteins, which in less than 2 years, enabled the IMP group to produce the first human ABC transporter structure in Oxford.

George (Georgina) Berridge

George Berridge has been interested in the scientific research of membrane associated and soluble proteins since 1989 and joined the SGC in 2004. Since 2007, through self-learning, George took over the running and maintenance of the mass spectrometry facility and continues to contribute to method development and standard assessment of the integral membrane proteins, soluble proteins and small molecules produced in house.

Rod Chalk

Rod Chalk gained his PhD in 1992 at Liverpool University. His early experience was in MALDI-TOF of bioactive peptides, nucleic acids and cryodetector MS of large proteins. More recently Rod has worked in proteomics and biopharmaceuticals using a variety of ESI instruments. Rod has held several roles within the biotechnology industry in the UK and abroad and joined the SGC in 2008.

Solenne Goubin

Solenne Goubin studied a 2 year Technical degree in Human Biology before joining the 3^rd year of the B.Sc. Biomedical Sciences at Greenwich University in Kent as an Erasmus student. She then worked in Pfizer R&D Sandwich for 2 years as cell culture scientist. In 2009, she joined the Biotechnology team at the SGC in Oxford. Solenne is interested in the biomedical field in general, and also in public health in developing countries.

Emilia Hapka

Emilia Hapka works on a part-time basis for the SGC as a research technician and is responsible for management and distribution of the SGC clone collection in addition to cloning of targets in the pipeline. Emilia is studying Graphic and Media Design in London, focusing on interactive design.

Grazyna Kochan

Grazyna Kochan has a broad range of experience in molecular virology, protein biology and immunology. After obtaining her PhD in molecular virology from the University of Gdansk in Poland, she did several post-doctoral placements at high quality scientific research centres, including Oxford University, where she studied the replicase activity of bluetongue virus and worked on the development of baculovirus expression vectors. Grazyna moved to Dr. Francisco Rodriguez’s group in the National Centre for Biotechnology (Madrid) where she continued with the study of RNA-binding proteins. She continued working on the development of viral vectors for immunotherapy at Prof. Mariano Esteban’s group at the CNB then joined the SGC in 2006. Grazyna has been working as a Senior Scientist/Team Leader with an interest in the structure and molecular mechanisms of metabolic enzymes and lately in the role of aminopeptidases in the immune response. Recently, she has been involved in developing methods for Biotechnology.

Pravin Mahajan

Pravin Mahajan received his M.Sc. in Microbiology at the University of Pune, India and his Ph.D. in drug-metabolising Cytochrome P450 system at De Montfort University, Leicester. Prior to his Ph.D. he worked in New Drug Discovery in the area of oncology and cardiovascular disease. Pravin joined the SGC in 2008 and has been working on high-throughput expression of human proteins in insect cells. Through collaboration, he is also working on Chromodomain-helicase-DNA-binding proteins with the aim of obtaining structural information.

Shubhashish Mukhopadhyay

Shubhash Mukhopadhyay completed his M.Sc. in Pharmacology and Biotechnology from Sheffield Hallam University in 2006. Previously, Shubhash worked at Reckitt Benckiser Plc., as a research analyst, working on chromatography of active pharmaceutical ingredients like paracetamol, phenylephrine and codeine and other process development projects in a cGMP environment, till 2008 and then worked at Oxford expression technologies Ltd. as a quality control assistant working on production and quality testing of recombinant bacmid DNA. 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.

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.

Claire Strain-Damerell

Claire Strain-Damerell joined the Biotech group in February 2011 and is currently involved in both the insect and bacterial HTP screening pipelines. Prior to joining the SGC, Claire undertook her DPhil project at the University of Sussex, characterising a redox-sensitive transcriptional repressor in Streptomyces coelicolor. Her background is predominantly DNA-manipulation and protein expression, as well as transcriptomics (microarrays, RT-qPCR) and DNA-protein interactions (ChIP-on-chip, EMSA, SPR).

Publications

Publications of the members of the new Biotechnology Group (2007-2011)

2011

· Wang AT, Sengerova B, Cattell E, Inagawa T, Hartley JM, Kiakos K, Burgess-Brown NA, Swift LP, Enzlin JH, Schofield CJ, Gileadi O, Hartley JA, and McHugh PJ. Human SNM1A collaborates with XPF-ERCC1 to initiate DNA interstrand cross-link repair. (2011) Genes Dev. 25:1859-1870.

· Berridge G, Chalk R, D’Avanzo N, Dong L, Doyle D, Kim J-I, Xia X, Burgess-Brown N, deRiso A, Carpenter LP and Gileadi O. HPLC separation and intact mass analysis of detergent-solubilised integral membrane proteins. (2011) Anal. Biochem. 410(2): 272-80. Epub 2010 Nov 17.

· Lucic B, Zhang Y, King O, Mendoza-Maldonado R, Berti M, Niesen FH, Burgess-Brown NA, Pike A, Cooper C, Gileadi O and Vindigni A. A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation. (2011) Nucleic Acids Res. Mar;39(5):1703-17. Epub 2010 Nov 8.

· Evans DM, Spencer CCA, Pointon JJ, Su Z, HarveyD, Kochan G, at al. Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide presentation as the mechanism for HLA-B27 in disease susceptibility. (2011) Nat. Genetics 43:761-767.

· Zhang Z, Kochan GT, Ng SS, Kavanagh KL, Oppermann U, Schofield CJ and McDonough MA. Crystal structure of PHYHD1A, a 2OG oxygenase related to phytanoyl-CoA hydroxylase. (2011) Biochem. Biophys. Res. Commun. 408:553-8.

· Kochan G, Krojer T, Harvey D, Vollmar M, Kavanagh KL, von Delft F, Wordsword P and Oppermann U. Crystal structure of the endoplasmic reticulum aminopeptidase-1 ERAP1 reveal the molecular basis for N-teminal peptide trimming. (2011) PNAS. 108:7745-50.

2010

· Savitsky P, Bray J, Cooper CDO, Marsden BD, Mahajan P, Burgess-Brown NA and Gileadi O. High-throughput production of human proteins for crystallization: the SGC experience. (2010) J. Struct. Biol. Oct;172(1):3-13.

· Kochan G,* Krojer TJ*, Leung IKH*, Henry L*, von Delft F, Claridge TD, Oppermann U, McDonough MA, Schofield CJ. Structural and Mechanistic Studies on γ-Butyrobetaine Hydroxylase Reveal an Unprecedented Rearrangement. (2010) Chem Biol. 22:1316-1324.

· Froese DS, Kochan G, Muniz JR, Wu X, Gileadi C, Ugochukwu E, Krysztofinska E, Gravel RA, Oppermann U and Yue WW. Structures of the human GTPase MMAA and vitamin B12-dependent methylmalonyl-CoA mutase and insight into their complex formation. (2010) J Biol Chem. 285:38204-38213.

· Mantri M, Krojer T, Bag EA, Webby CE, Butler DS, Kochan G, Kavanagagh KL, Oppermann U, McDonough MA and Schofield CJ. Crystal Structure of the 2-Oxoglutarate- and Fe(II)-Dependent Lysyl Hydroxylase JMJD6. (2010) J. Mol. Biol.401:211-222.

· Healy S, McDonald MK, Wu X, Yue WW, Kochan G, Oppermann and Gravel RA. Structural impact of human and Escherichia coli biotin carboxyl carrier proteins on biotin attachment. (2010) Biochemistry 49:4687-94.

· Froese DS, Healy S, McDonald M, Kochan G, Oppermann U, Niesen FH and Gravel RA. Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder. (2010) Mol. Genet. Metab. 100:29-36.

2009

· Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Müller S, Knapp S. (2009) Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell 136(2):352-63.

· Pike AC, Shrestha B, Popuri V, Burgess-Brown N, Muzzolini L, Costantini S, Vindigni A, Gileadi O. (2009) Structure of the human RECQ1 helicase reveals a putative strand-separation pin. PNAS 106(4):1039-44.

· Grundler R, Brault L, Gasser C, Bullock AN, Dechow T, Woetzel S, Pogacic V, Villa A, Ehret S, Berridge G, Spoo A, Dierks C, Biondi A, Knapp S, Duyster J, Schwaller J. Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration. J Exp Med. (2009) Aug 31;206(9):1957-70. Epub 2009 Aug 17.

· Pilka ES, Niesen FH, Lee WH, El-Hawari Y, Dunford JE, Kochan G, Wsol V, Martin HJ, Maser E and Oppermann U. Structural basis for substrate specificity in human monomeric carbonyl reductases. (2009) PLOS One 10:e7113.

· Picaud SS, Muniz JR, Kramm A, Pilka ES, Kochan G, Oppermann U, Yue WW. Crystal structure of human carbonic anhydrase-related protein VIII reveals the basis for catalytic silencing. (2009) Proteins 76:507-1.

· Bunkoczi G, Misquita S, Wu X, Lee WH, Rojkowa A, Kochan G, Kavanagh KL, Oppermann U and Smith S. Structural basis for different specificities of acyltransferases associated with the human cytosolic and mitochondrial fatty acid synthases. (2009) Chem. Biol. 16:667-75.

· Kochan G, Pilka ES, von Delft F, Oppermann U and Yue WW. Structural snapshots for the conformation-dependent catalysis by human medium-chain acyl-coenzyme A synthetase ACSM2A. (2009) J. Mol. Biol. 22:997-1008.

2008

· Burgess-Brown N, et al., Codon optimization can improve expression of human genes in Escherichia coli: a multigene study. Prot. Expr. Purif. (2008) 59(1):94-102.

· Gileadi O, Burgess-Brown NA, Colebrook SM, Berridge G, Savitsky P, Smee CE, Loppnau P, Johansson C, Salah E, Pantic NH. (2008) High throughput production of recombinant human proteins for crystallography. Methods Mol Biol. 426:221-46.

2007

· Elkins JM, Papagrigoriou E, Berridge G, Yang X, Phillips C, Gileadi C, Savitsky P, Doyle DA. Structure of PICK1 and other PDZ domains obtained with the help of self-binding C-terminal extensions. (2007) Protein Sci. Apr;16(4):683-94.