Protein Production

Instruct offers a wide range of expression systems for recombinant proteins including Bacterial, Baculovirus, Cell-free, Mammalian and Yeast. Our techniques allow for expression of challenging proteins along with expert protein purification systems.

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Instruct has 7 centres offering Protein Production across Europe. Navigate the map and click on the pins to discover centres near you.

Protein Production Details

Bacterial Expression

Bacterial hosts are hosts of choice for the production of recombinant proteins and protein complexes. This is largely due to the ease of handling and genetically modifying these hosts. In addition, the short doubling time of bacteria enables fast production of samples using parallelised and robotised approaches. Furthermore, the large biomass and high expression yields that can be obtained with bacterial hosts generally enables the production of sufficient amounts of samples for structural studies.

Baculovirus/ insect cell expression (BEVS) 

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 have become available, and the BEVS is now accessible also 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.

Cell-free expression 

Cell-free expression is in particular suitable for the production of difficult proteins such as membrane proteins, toxins, complexes or peptides. Expression is performed in cell-free extracts of different 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 incubation times usually do not exceed 12-16 hrs. With optimized production protocols, several mg of protein in one ml of reaction can be obtained.

Mammalian expression

Production of functional mammalian proteins in prokaryotic or insect cells can be hindered by the lack of the correct folding machinery and post translational modifications. To overcome this problem the mammalian expression platform proposes to express intra cellular and secreted proteins and protein complexes in several mammalian cell lines with a number of expressions systems. In addition to the commercially available expression vectors, specific expressions systems developed on the platform will be available to the user. The platform will offer a facility with two levels of complexity. The first level will be to produce recombinant proteins with vectors provided by the user. The second level will be the construction and screening of several expression vectors to optimize the production of functional and soluble protein and protein complexes.

Large scale biomass production

The Instruct facility also supports the routine production of recombinant mammalian proteins by mammalian cell lines or the Baculovirus system in 50 L scale. Both secreted as well as intracellular proteins can be produced in up to 200 L of cell culture by a perfusion process or semi-continuous fermentation.

Stable CHO cell line development by RMCE

A unique and fast method to create stable, glycosylation mutant CHO production cell lines using a high producer master cell line and site specific recombination allows faster access to difficult to express mammalian glycoproteins (Wilke et al., Protein Science, Prot Science 19 (2010), p1264). The method is based on cloning a gene of interest into an exchange vector and subsequent specific integration of the expression cassette into a preselected high producer master cell line. The resulting production cell lines can be generated in two to four months, which is substantially faster than by classical integration techniques. The resulting cell lines allow the production of mg amounts of mammalian protein. The performance and stability of new producer cell lines has been tested in batch and continuous fermentation. This strategy increases the throughput of producing difficult target proteins in the mammalian stable CHO expression system.

Yeast expression 

Yeast is an organism which is well-suited for the expression of recombinant eukaryotic proteins or the production of endogenous complexes. Advantages include the low cost of growing material, robustness of the system, easy genetic manipulation, and the presence of endogenous eukaryotic-type protein modification and chaperone systems 

Library methods for soluble expression of challenging proteins

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.

Using DNA deletion or fragmentation protocols, libraries of tens of thousands of constructs of a single gene are synthesised and 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. For a review of this subject, please see: Yumerefendi H, Desravines DC, Hart DJ (2011). Library-based methods for identification of soluble expression constructs. Methods 55(1):38-43.

Purification: chromatographic system 

Proteins of interest are usually purified from endogenous and recombinant sources using different cellular organisms or cell-free systems. Protein purification consists of a series of separation steps intended to isolate a single type of protein from a complex mixture. Separation steps may exploit differences in protein size, physicochemical properties, binding affinity and biological activity. High-performance purification is performed on preparative or analytical scale columns using chromatographic systems. Usually the proteins are detected as they are eluted from the column by their absorbance signal. Preparative purifications aim to produce a relatively large quantity of purified proteins (> 1 mg) for subsequent structural and functional studies. Analytical purifications produce a relatively small amount of proteins (0.1 - 1 mg) for a variety of research purposes and are commonly used to set up a purification protocol. Micro-purification is a technique adapted to the isolation of endogenous complexes produced in very low amount (< 0.1 mg) and difficult to express macromolecular complexes. To evaluate expression levels in multiple small test cultures and to screen for soluble or mutagenized protein constructs, a partial purification using a single affinity step may be performed on robotic systems.

Purification: Lysis system 

Instruct offers extraction of endogenous or recombinant proteins and protein complexes from cells or tissues and their recovery in solution for further analysis. For purification and subsequent research purposes, the protein has to be brought into solution by breaking the tissue or cells containing it. Several methods of cell lysis are commonly used to achieve this : mechanical disruption, sonication using high frequency sound waves, liquid homogenization and freeze/thaw cycles. For example, the beating of glass beads on cells crush cell walls, cell suspensions are sheared by forcing them through a narrow space and high pressure/sudden depressurization disrupt cells. The choice of the lysis method depends on the type of cells (bacteria, yeast, mammalian cells, etc), the volume of cell suspension and the fragility of the proteins to be recovered. The composition of the lysis buffer is also crucial to maintain the proteins of interest in the soluble fraction after cell breakage.