Prokaryotic Expression Team – University of Copenhagen

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CPR > Research > Protein Structure & Function Program > Protein Production Platform > Prokaryotic Expression...

Prokaryotic Expression Team

The Prokaryotic Expression Team consists of technicians Khalid Pardes, Havva Koc, Matteo Bellucci and Motiejus Melynis, and is headed by Academic Coordinator Andrea Vala.

The primary objective of the prokaryotic expression team is to express and purify proteins from E. coli for the research groups at CPR. This process typically includes cloning, expression optimization, large-scale expression, and purification.

Expression constructs are created using ligation-independent cloning (LIC) fusing the proteins with an affinity tag such as 6xHis, Twinstrep, or glutathione S-transferase (GST). All affinity tags can be removed by TEV protease cleavage, leaving a near-native protein. All expression constructs are confirmed by sequencing.

Optimization of protein expression can be achieved by varying parameters including: expression strain, media, duration and temperature of expression, and lysis method. Currently, expression and solubility are evaluated using a standard panel of 24 conditions, and the optimal condition is selected for large-scale expression.

Large-scale expression is performed using either shaker incubators or the LEX bioreactor. Standard protein purification is performed using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). For custom purifications, ion-exchange chromatography (IEC) can also be applied. Purity is assessed using SDS PAGE, and identity is confirmed using mass spectrometry.

The team also offers training for CPR researchers in protein expression and purification.

Recent publications

Säll A, Walle M, Wingren C, Müller S, Nyman T, Vala A, Ohlin M, Borrebaeck CAK, Persson H. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays. Protein Eng Des Sel. 2016, accepted 

Kibat J, Schirrmann T, Knape MJ, Helmsing S, Meier D, Hust M, Schröder C, Bertinetti D, Winter G, Pardes K, Funk M, Vala A, Giese N, Herberg FW, Dübel S, Hoheisel JD. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality. N Biotechnol. 2016,33 (5 Pt A):574-81

Starnes LM, Su D, Pikkupeura LM, Weinert BT, Santos MA, Mund A, Soria R, Cho YW, Pozdnyakova I, Kubec Højfeldt M, Vala A, Yang W, López-Méndez B, Lee JE, Peng W, Yuan J, Ge K, Montoya G, Nussenzweig A, Choudhary C, Daniel JA. A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex. Genes Dev. 2016; 30(2):149-63.

Christensen LC, Jensen NW, Vala A, Kamarauskaite J, Johansson L, Winther JR, Hofmann K, Teilum K, Ellgaard L.  The human selenoprotein VCP-interacting membrane protein (VIMP) is non-globular and harbors a reductase function in an intrinsically disordered region.  J Biol Chem. 2012; 287(31):26388-99.

Vernet E, Sauer J, Andersen A, Jensen KJ, Voldborg B.  Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation.  Anal Biochem. 2011; 414(2):312-4.

Vernet E, Kotzsch A, Voldborg B, Sundström M.  Screening of genetic parameters for soluble protein expression in Escherichia coli.  Protein Expr Purif. 2011; 77(1):104-11.

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; 20(3):597-609.