Instruct's services include protein expression, cloning and high throughput expression, and protein purification. Our techniques allow for expression of challenging proteins along with expert protein purification systems.
Instruct has 9 centres offering Protein Production across Europe. Navigate the map and click on the pins to discover centres near you.
Instruct offer protein expression in E.coli, BEVS, mammalian cells and cell-free extracts:
The gram negative bacteria, E. coli is the first choice expression host for the production of recombinant proteins and protein complexes from prokaryotes and can be used to produce eukaryotic proteins particularly the structural/functional domains of larger proteins. The advantages of using E. coli include the ease of handling, short cell doubling time leading to rapid production of samples and the low cost of scaling up to multi-litre volumes to produce high yields of proteins. Most laboratories have access to E. coli expression technology. Instruct-ERIC Centres can help with optimisation of expression using high throughput cloning and screening platforms (see below).
The baculovirus expression vector system (BEVS) is a powerful eukaryotic method to produce proteins and protein complexes. BEVS uses a recombinant baculovirus carrying the heterologous genes of interest, to infect insect cell cultures provided as monolayers or suspension. BEVS has become particularly attractive for protein research as it combines the ability to produce difficult-to-express, eukaryotic proteins with high yields and often authentic processing (PTMs) with simple cultivation needs without particular safety precautions. Important technological advances have greatly improved upon the original procedures for the generation of recombinant baculoviruses which were time consuming and required specialised expertise. Streamlined and cost-effective operation procedures for baculovirus-based expression are available at several Instruct-ERIC Centres, ensuring that the BEVS is accessible to non-specialist users. Within the Infrastructure, new technologies have been developed and implemented which enable efficient and rapid production of large multiprotein complexes with many subunits, in the quality and quantity required for high-resolution structural and functional studies.
Production of functional mammalian proteins in either E. coli or insect cells can be hindered by the lack of either the correct folding machinery or post translational modifications or both. Expression of proteins in mammalian cells can be used to overcome this problem. Both viral (e.g. using vaccinia viruses) and non-viral delivery systems are available at different Instruct-ERIC Centres for mammalian cell expression.
Cell-free expression in a coupled transcription-translation system is particularly suitable for the production of difficult proteins such as membrane proteins, toxins, complexes or peptides. Expression is performed in cell-free extracts of different cellular origins and reactions can be supplemented with a variety of compounds such as ligands, cofactors, inhibitors, chemicals, detergents or lipids. Reaction volumes are small in the range of few ml and with optimized production protocols, several >/= 1 mg of protein in a 1 ml reaction can be obtained. The main disadvantage of cell-free technology is the cost of preparing the cell extracts. Expertise and technology is available within Instruct-ERIC.
Instruct provides two different types of platform for identifying or optimising expression constructs:
The design, cloning and expression screening of multiple constructs for a given protein target has become a standard approach to optimising protein production. Semi-automated workflows enabling high throughput vector construction by ligation-independent cloning combined with expression testing in different systems (bacteria, mammalian and insect cells) have been established in a number of Instruct-ERIC Centres. Typically, 5-10 different versions of a target are evaluated with multiple targets handled in parallel in SBS plate formats (24 or 96-wells). Constructs include both sequence variants and vector configurations that incorporate different fusion tags for detection and purification.
Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, Rahman N, Stuart DI, Owens RJ. (2007). A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.Nucleic Acids Res. 2007; 35(6):e45. PMID: 17317681
Structural biologists often work on proteins that lack accurate domain annotations. When the full-length protein cannot be expressed and a domain-focused approach is necessary, problems arise since it is unclear how to design high yielding, soluble expression constructs. Some proteins have little or no sequence similarity to others and this prevents domain identification using multiple sequence alignments. More often, some functional annotation exists e.g. from mutagenesis or deletion studies, but these regions do not define well the structural boundaries. Even when a soluble construct is obtained, disordered extensions may confound crystallisation attempts.
The ESPRIT method users DNA deletion or fragmentation protocols to generate random libraries of tens of thousands of constructs of a single gene that are screened for rare clones that produce folded soluble domains. These are identified empirically and require no knowledge of the domain content of the target. The systems here can address single proteins, or simple complexes, and can be used to identify domains de novo, or to improve existing domains for crystallisation.
Instruct-ERIC offers purification of endogenous or recombinant proteins and protein complexes including membrane proteins. Semi-automated chromatography systems are used to perform preparative purifications combining affinity capture with size exclusion and/or ion exchange separation methods.
