Protein expression on demand

This article looks at a novel approach to meeting protein research needs

SINCE the entire human genome was first sequenced in 2000, the decreasing costs and increasing availability of genetic information has shifted the focus of research towards functional genomics, exploring the biological significance of the native product of any given gene. The numerous splice variants and post-translational modifications that exist for many genes have led to a significant expansion in the field of proteomics. Understanding the structure and function of these proteins often requires isolation of the purified gene product to allow in-depth analysis of protein structure, using techniques such as crystallography and NMR. In addition, functional studies based on in vitro biochemistry and mutagenesis strategies can require large quantities of purified proteins to ensure reliable results.

Effective protein expression and purification are key elements in proteomic research, yet traditional cloning and expression techniques have only been applied on a piecemeal basis, unsuited to large scale protein expression. Recent advances in cloning techniques, combined with new hardware technologies, have resulted in the development of high throughput strategies allowing highly predictable, parallel expression and purification of large numbers of proteins. Many of these approaches are suited to automation on liquid handling workstations, further increasing throughput and improving reproducibility.

The University of York has taken a novel approach to meeting its protein research needs, creating a dedicated facility for high throughput gene cloning and protein expression. A joint venture of the Departments of Biology and Chemistry, the High Throughput Expression Laboratory (HiTEL) uses modern cloning and expression techniques, in combination with state-of-the-art technology, to provide a comprehensive protein production service. To meet the throughput demands of supporting a number of university laboratories, external institutes and industry laboratories, the group relies on automation of its gene cloning and protein expression workflow using a Freedom EVO 200 platform from Tecan. This flexible workstation is used to automate as much of the laboratory’s work as possible, ensuring reliable gene cloning and protein expression. To maximise throughput and efficiency, the HiTEL’s Freedom EVO system has been customised to match the specific requirements of the laboratory’s workflow, and this increased capacity has allowed the laboratory to participate in a number of international research collaborations in addition to its service role, including the SPINE2-COMPLEXES work program and the Bacillus Systems Biology project.

The HiTEL uses bespoke protocols to offer fully customised cloning and expression from various bacterial, parasite, animal and plant genomes. The group’s parallel cloning strategy is based on semi-automated PCR technologies developed by the Protein Production and Structural Biology laboratories at the university. Target DNA sequences can be cloned into a range of specifically engineered expression vectors, using either ligation independent cloning (LIC) or InFusion cloning, with primers that are designed in house with the aid of purpose-built software. Working in a microplate format, this six stage process allows up to 96 targets to be cloned in parallel, and includes built-in verification of results and quality control steps. Figure 1 shows the semi-automated cloning pathway used in the laboratory, describing both cloning of the target DNA and transformation of the purified plasmid into an E. coli expression strain for small scale protein production. All constructs used in this process encode either an N-terminal or C-terminal histidine (His) tag, used for expression screening and affinity purification. The HiTEL has a library of expression vectors to offer a wide range of purification tags, including green fluorescent protein (GFP), glutathione-S-transferase (GST), maltose binding protein (MBP) and IM9. This process requires integration of many additional devices onto the laboratory’s Freedom EVO workstation, including an automated thermal cycler to allow complete automation of target gene amplification and purification on the platform as well as automatic normalisation of sample concentrations. Subsequent ligation into plasmid vectors and transformation into suitable cell lines is also performed by the workstation, minimising variation between samples and reducing the risk of operator errors.

Figure 1:  Semi-automated cloning pathway used by HiTEL

The laboratory also offers automated co-expression of protein complexes, using the LIC Duet vector system. This technique allows two genes to be cloned in series in the same plasmid. The first protein is expressed with a His tag (similar to individual target expression), while the second is in the native form. His-chromatography then yields either the complexed proteins, or the His-tagged protein only, offering a simple method for determining the efficiency of complex formation.

In addition to high throughput bacterial expression, the group has developed an automated eukaryotic expression system in a 24-well microplate format. Based on baculovirus vectors, this method can be used with intracellular expression, target protein secretion (directed by a gp64 secretion signal) or co-expression strategies. Expression testing is performed using suspension adapted insect cells in a 24-well bioreactor system, followed by SDS-PAGE analysis and immunodetection.

Following successful cloning, protein expression screening begins with transformation of purified plasmids into E. coli expression strains (BL21, Rosetta2, and B834 pLysS) (Figure 2). Positive colonies are induced with IPTG or transferred to auto-induction media to growth. Cell lysis is achieved using either sonication or a lysis reagent. A solubility screen is then achieved using filter-based technology and a vacuum manifold, followed by rapid analysis of the resulting fractions using SDS-PAGE and immunodetection techniques.

Figure 2: Semi-automated expression screening performed by HiTEL

These small scale trials are used to establish the optimum incubation temperature and induction method for expression of the target protein. Next, the process is scaled up to 0.5 L cultures to produce large amounts of protein. Following incubation, these large cultures are centrifuged, and the resulting cell pellets are harvested and lysed using a cooled cell disruptor. Supernatants are filtered and purified on a Ni2+-chelating column, followed by size exclusion chromatography (SEC) at 4ËšC. This semi-automated process allows several proteins to be co-purified in a single experiment, improving laboratory throughput as well as helping to make protein expression very predictable and reproducible. As a result, HiTEL staff have a high level of confidence in the amount of protein that can be produced from any given gene or protocol.

In addition to 0.5L cultures, the group has demonstrated that protein expression can be successfully scaled up to allow large volume production, with cultures of up to 50 litres readily achievable in bacterial fermenters. Similarly, eukaryotic systems can be scaled to 10 litre cultures in single use bioreactors, which can generate approximately 1mg of secreted proteins per litre. To complement this work, the laboratory offers an expression characterisation service to customers who are developing large scale bioproduction processes. Screening up to eight samples per experiment, this service allows 24 variables of choice to be investigated, including a range of expression cell lines, induction systems, temperatures and time courses. Expression is confirmed by immunodetection of the total and soluble cell extracts, providing valuable data to accelerate bioprocess development.

The HiTEL’s automated protocols are continually updated to incorporate the latest cloning technologies and improve laboratory workflow. As part of this, the group has recently developed a completely automated process to accelerate expression screening including all steps from solubilisation of the protein through to dot blotting and immunodetection analysis (of total and soluble fractions) using an integrated microplate reader. This new screen allows up to 80 samples to be assayed in parallel, and gives an indication of the expression level which could be expected on scale-up.

High throughput gene cloning and protein expression strategies are essential to the future expansion of functional genomics research and allow the development of rapid proteomics solutions for large scale, systematic gene product analysis. Automation of cloning and expression techniques improves the reliability of these methods, offering greater reproducibility by eliminating the inherent variability and errors associated with manual techniques. Automated platforms, such as the Freedom EVO system, contribute additional value by increasing walkaway time for labour intensive laboratory processes, allowing staff to perform other tasks, ultimately increasing the rate of discovery and bioprocess design.