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Protein Services

Tampere University
Faculty of Medicine and Health Technology
Kauppi Campus
Arvo Ylpön katu 34, 33520 Tampere
 
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Protein Services

Introduction

The facility offers services in protein expression, purification and characterization. The currently provided protein expression hosts (bacterial, insect and mammalian expression) cover a broad range of expression and post-translational modification requirements for a variety of life-science and industry needs. The produced recombinant proteins may be used in e.g. structural biology, protein-ligand and protein-protein interaction studies, and in the development of diagnostics.

Protein Expression Services

Expression vectors

The used expression vectors can either be provided by the customer or designed, case-by-case, by the Protein Service. The used vector backbone, included domains, fusion tags, linkers and protease cut-sites are selected based on the project needs.

E. coli protein expression

The Service carries a selection of E. coli strains for the expression of different types of proteins. For unknown proteins, we typically perform a small-scale expression test to confirm successful protein expression and to optimize expression parameters. After positive results, production-scale expression is performed using shake-flask cultures or small-scale fermentation system. 

Protein Service offers protein production in bacterial hosts in up to 4.5 l batches using Labfors Infors 3 fermentor system. The accurately controlled growth conditions of the system enable higher cell densities and better product quality than what is achieved using conventional shake-flask cultures.

Insect cell expression using baculovirus system

The provided baculovirus expression service is based on the Bac-to-Bac system. Customer-provided or service-made donor plasmids are used to prepare 3-6 bacmids and the correct transposition of the gene-of-interest confirmed by PCR. Confirmed clones are transfected into the Sf9 or HighFive insect cells. Successful expression of the protein is typically confirmed at the level of secondary virus (P2, 50 ml culture volume) using Western blotting. Validated virus stocks are stable for up to one month and can be used both for further optimization of expression conditions and expression scale-up to 500 ml - 4.5 liter media volume.

Mammalian protein expression

The provided mammalian expression service is based on CHO and HEK 293 cell lines and covers both transient and stable protein expression. For most small-scale protein expression projects, transient expression provides the best balance between protein yield and resource use. For previously unknown proteins, a 25 ml pilot culture is initially used to confirm protein expression and solubility. Expression scale-up is possible up to 5-liter scale using parallel shake-flask cultures or Cytiva Wave bioreactor system.

 

Protein Isolation, Purification, and Characterization

Host cells and expression media are separated by centrifugation or filtration. For the purification of intracellular and periplasmic proteins, the cells are lysed using Emulsiflex high-pressure homogenizator, a probe sonicator or enzymatic methods. Protein isolation, purification and final polishing are typically done using different chromatographic methods (IMAC, IEX, SEC) ran using automated FPLC systems. When purification tag removal is necessary, specific proteases (e.g. TEV and 3C) can be used. Based on the project needs, quality of the final protein preparation can be evaluated using several methods, including analytical gel filtration, SDS-PAGE, 2D electrophoresis, dynamic light scattering, mass photometry, calorimetric methods and biosensor systems. The final product is provided to the customer either as a lyophilized powder or flash-frozen liquid in selected buffer system.

The Resources tab provides a brief summary of the tools and biophysical characterization instrumentation available.

MET Core Facilities and Services