Expression and Purification of Proteins and Complexes

The purity and homogeneity of the protein/complex sample is one of the most important factors that influence the success of subsequent crystallization experiments. At the Crystallisation Factory we have all the expertise and key equipment needed for the expression and purification of soluble proteins and macromolecular complexes:

• HPLC (ÄKTApurifier™): Liquid chromatography system designed for fast and reliable separations of peptides, nucleic acids, oligonucleotides and proteins. The HPLC runs all techniques from μg to mg scale at flow rates 0.001 m/min - 10 ml/min and pressures 0-25 MPa and incorporates many features to minimise preparation and run times and maximise reliability and reproducibility of results. Additionally, it allows on-line UV-detection of up to three wavelengths simultaneously to detect contaminants at higher sensitivity wavelengths (e.g. 280/214/254 nm).

• FPLC (ÄKTA prime Plus™): The low-pressure FPLC system (1 MPa max with flow rate of 0.1-50 ml/min) is primarily designed for protein purification. It contains an UV monitor (254 and 280 nm), a conductivity meter (0.001-999.9 mS/cm), a gradient maker/mixer and an online pH monitor. The system can be operated manually (real-time), with pre-programmed protocols or custom/user-defined protocols.

• Dynamic Light Scattering: The Zetasizer Nano instrument (Malvern Instruments) can be used to measure the particle size, zeta potential and molecular weight of molecules in solution over a wide range of concentrations with a precise control of temperature for reproducible, repeatable and accurate measurements.

• `Air-driven’ Ultracentrifuge: Easy-to-operate table-top centrifuge that reaches top speed (100.000 rpm) in as little as 30 seconds using very small sample amounts (tubes of 150µl maximal capacity).

• Protein purification columns: A wide range of protein purification columns for analytical and preparative purification procedures. We have columns (affinity, desalting, ion exchange, hydrophobic resins and size exclusion) of different sizes and features to answer all user requirements for their use in different purification set-ups.

• Library of plasmids for the expression and purification of heterologous proteins: We offer bacterial expression vectors for the heterologous production of single proteins and protein complexes through alternative expression protocols (single expression plasmids and bi- and multi-cistronic vectors for co-expression strategies).

Technological services

It is possible to choose from 5 main types of services ranging from the simple production of proteins/complexes until its complete purification to obtain a homogeneous sample.

1. Protein Expression Test (prokaryotic systems):  Determination of the best conditions for protein expression including quantification the soluble fraction and pellet using 27 conditions (3 E. coli strains x 3 inductor conc. x 3 Tª).

2. Purification of Proteins and Complexes: It includes the purification of proteins (homogeneous and pure sample) and/or macromolecular complexes (pure, homogeneous and good stoichiometry) by the use of chromatographic techniques (affinity chromatography, ion exchange, hydrophobic resins or molecular exclusion). The standard purification includes a chromatography column; but additional columns can be requested.

3. Complete Pack of Expression and Purification: The combo pack includes all the necessary steps necessary for the production and purification of a protein from 2 L of culture. There are three options available depending on the number of purification steps: the expression plus a purification step, the expression with two purification steps and the expression with three steps of purification.

4. TEV Fusion Protein Cleavage: Elimination of proteins or fusion tails.

5. Thermofluor: Search of the optimal solubilisation buffer.