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Biotechnology

The Biotechnology group
Group Site: 
toronto
Group Leader: 

Susanne Graslund

Group Info

Research Areas

The Biotechnology team

The Biotechnology team is responsible for running a high-throughput generic protein production process. The group performs high-throughput cloning in 96-well format, typically 10 constructs per target, into a range of vectors for expression in different hosts (primarily E.coli and Insect cells) and with different tags. Most constructs include a hexahistidine tag enabling affinity chromatography purification and a protease cleavage site so that the tag can be removed after protein purification. The cost-efficient Ligase-independent cloning (LIC) procedure used is well suited for a high-throughput format. Small-scale cloning to produce specific mutants for various projects is also carried out when needed

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Expression

Small-scale expression screening in 96-well format in E.coli is either carried out by the Biotech team or by the other teams. Promising constructs are scaled up and grown in 1- or 2-Litre Terrific broth cultivations in a Large-scale expression (LEX) system.

The Biotech team also does a lot of protein production in Insect cells using the Baculovirus expression system for intracellular or secreted expression of target proteins. Expression screening is performed in 24-well format and promising candidates are being scaled up in either shake flasks or in the Wave Bioreactor depending on the volume. 

Purification

The generic protein purification method usually includes a two-step purification protocol (IMAC+GF) run on a 4-module ÄKTA Xpress which can accomodate up to 16 samples in parallel. The purity and quality of the proteins is analysed by SDS-PAGE before they are concentrated All batches are also analysed by Mass spectrometry to verify protein identity and identify possible inhomogeneities or modifications.

The Epigenetics binders project

The Biotech team takes part in a project to produce renewable antibodies to target proteins involved in Epigenetic mechanisms in collaboration with LIFE Technologies and several research groups that perform binder selections and cell-based validation experiments. The project tasks performed within the Biotech team includes cloning of expression constructs for antigen production, production of in vivo biotinylated antigen batches, re-cloning of selected binder candidates, production of in vivo biotinylated binder candidates for secondary validation, re-cloning of validated binders into IgG conversion vectors, production of IgG-like molecules using transient expression in mammalian cells and purification of IgG-like molecules.

Group Members
Susanne Gräslund, PhD

Dr. Gräslund did her doctoral training at the Dept. of Biotechnology, KTH with Prof. Stefan Ståhl. After her dissertation in 2002 she worked for three years at Biovitrum AB in the Target Expression & Purification section. In March 2005, Susanne joined SGC Stockholm, heading the Biotechnology team which was responsible for the generic protein production pipeline. She was then recruited to the Toronto site as Principal Investigator for the Biotechnology team from September 2011.

Peter Loppnau, MSc
Yanjun Li, MSc

Supported by Word Health Organization, Yanjun Li did her visiting scholar training in Baylor College of Medicine for 2 years. She then worked 6 years in Stem Cell Biology Lab directed by Dr. William Stanford (IBBME, U of T), responsible for establishing gene trapping and gene targeting vectors and high throughput trapped mouse ES screenings. She transferred to Dr. Michelle Bendeck lab (LMP, U of T) in 2005 assisting supervisor in grant application, mouse protocol renewal, routine lab maintenance and transgenic mouse lines maintenance. Yanjun joined SGC Biotechnology Team as a cloner in May 2007.

Alma Seitova, PhD

Dr. Seitova obtained her PhD degree and postdoctoral position the next following years at the Dept. of Molecular Biology, Academy of Science, Moscow. After that she spent a few years at Centre "Bioengineering", (RAS).  In 2000, she joined the Cardiovascular Biology Department at OMRF, USA, where a therapeutic r-protein, Xigris, was produced using HEK293 cells. Dr. Seitova's project included production of the recombinant protein involved in blood coagulation cascade using stable and transient transfection of mammalian cells. Since 2005 she works in the Biotechnology group at SGC, currently in charge of the Eukaryotic Expression Platform.

Hao He, PhD

After three years as a general surgery doctor, Dr. Hao He did his doctoral training at the Dept. of Biochemistry and Molecular Biology, Medical school Beijing University in China. After his dissertation in 1995 he worked for three years as a post-doc at the Academy Military Medical Institute in Beijing. In Sep 1998, Dr. He moved to Sweden and worked in Department of Clinical Physics at Uppsala University for three years as a research fellow. In 2001, Dr. He moved to Canada and worked for 2 years at the Kidney Research Centre, Ottawa general Hospital. From 2003 to 2006, he worked in Dept. of Transplantation at the Toronto General Hospital for fgl2 and CD200 protein expression using Baculovirus/Insect cells and CHO cells. He then joined SGC Toronto where he is working with Baculovirus expression system in Insect cells.

Nan Zhong, PDF
Elena Dobrovetsky, MSc

Elena Dobrovetsky received her Master’s degree in Structure Determination of Biological Macromolecules by X-Ray Crystallography from Israel Technion, Schulich Faculty of Chemistry. She was then recruited to Prof.  Aled Edwards’s lab in August 2002  to work on a membrane proteins project. In 2007 she joined the Biotechnology group at SGC.

Mani Ravichandran, MSc

Mani Ravichandran did her Master's degree in Biochemistry with specialization in Clinical Biochemistry at Bharathiyar University, India. She worked as a lecturer in the Faculty of Biochemistry, Perundurai Medical College and Research Centre, India before joining the Biotechnology team at SGC Toronto in 2004. She is responsible for preparing in-house protein crystallization screens, protein purification and crystallization.

Publications

1.       Colwill K; Renewable Protein Binder Working Group; Gräslund S. A roadmap to generate renewable protein binders to the human proteome. Nat Methods. 2011 May 15;8(7):551-8. doi: 10.1038/nmeth.1607.

2.       Kotzsch A, Vernet E, Hammarström M, Berthelsen J, Weigelt J, Gräslund S, Sundström M. A secretory system for bacterial production of high-profile protein targets. Protein Sci. 2011 Mar;20(3):597-609. doi: 10.1002/pro.593

3.       Pershad K, Pavlovic JD, Gräslund S, Nilsson P, Colwill K, Karatt-Vellatt A, Schofield DJ, Dyson MR, Pawson T, Kay BK, McCafferty J. Generating a panel of highly specific antibodies to 20 human SH2 domains by phage display. Protein Eng Des Sel. 2010 Apr;23(4):279-88. Epub 2010 Feb 17

4.       Mersmann M, Meier D, Mersmann J, Helmsing S, Nilsson P, Gräslund S, Structural Genomics Consortium, Colwill K, Hust M, Dübel S. Towards proteome scale antibody selections using phage display N Biotechnol. 2010 May 31;27(2):118-28. Epub 2009 Oct 31

5.       Uhlén M, Gräslund S, Sundström M. A pilot project to generate affinity reagents to human proteins. Nat Methods. 2008 Oct;5(10):854-5.

6.   Gräslund S, Nordlund P, Weigelt J et al. Protein production and purification. Nat Methods (2008) Feb;5(2):135-46.

7.   Gräslund S, Sagemark J, Berglund H, Dahlgren LG, Flores A, Hammarström M, Johansson I, Kotenyova T, Nilsson M, Nordlund P, Weigelt J. The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. Protein Expr Purif. (2008) Apr;58(2):210-21.

Contact

Email: susanne [dot] graslund[at]utoronto [dot] ca

Phone: 416-978-7842