Large-scale expression

Clones that score sufficiently high in the small-scale expression analysis can be sent for large scale expression and purification. Here, we use a large-scale expression (LEX) system developed in SGC Toronto but made publically available by Harbinger Biotech. In principle, the LEX is a temperature-controlled water-bath with 24 separate air supplies adapted to fit into regular 1- or 2-liter glass flasks. Spargers attached to the air hoses fulfill the dual purpose of aeration and agitation, thus eliminating the need for bulky shaking platforms. The LEX is a crucial piece of hardware that enables us to perform multiple and parallel large-scale cultivations (up to 1.5 liters per flask) in a very small laboratory foot print.

Sample preparation for downstream purification include harvest, cell lysis by sonication, lysate clarification by centrifugation and a final 1.2 uM filtering step. Protease inhibitors and DNAse are added during cell pellet resuspension in order to minimize target protein degradation and ease sample handling, respectively. All steps are performed rapidly and on ice. We have had great success with using a HEPES-based buffer containing 0.3-0.5 M NaCl, 10% glycerol and 0.5-2 mM TCEP at a pH of 7.5.

Target protein purification is typically a two-step affair involving an initial IMAC purification followed by size exclusion chromatography (SEC or ‘gel filtration’). We routinely use the highly automated ÄKTAxpress systems for all purifications and while this certainly restricts the techniques at our disposal, we have found that the massive increase in throughput that we gain from these instruments is far more valuable to our pipeline than alternative purification strategies. Each ÄKTA module can accomodate up to four samples and as we currently have four at our disposal, we can purify up to 16 proteins in parallel. However, as previously mentioned, we are not able to perform gradient elution, ion-exchange, or TEV-cleavage on a routine basis. Similarly, any requests for specific buffer compositions will only be catered to in certain cases.

Immediately following purification, GF-fractions will be pooled and the protein will be concentrated as per request. Sample purity will be assessed on SDS-PAGE and the exact size of the protein will be determined using mass spectrometry. All protocols and results will be available on the LIMS system for your reference.

While we do appreciate that our approach may not coincide with your views on how to purify a specific protein, we refer to the massive amount of data that supports this generic approach towards protein production. Globally, the SGC has submitted close to 2000 high-resolution protein structures (http://www.thesgc.org/structures) into the Protein Data Bank and while all of these proteins were certainly not produced using the same protocol, the vast majority of them went through a pipeline similar to ours. Naturally, we will try to accommodate any special requests that you might have - feel free to talk to us!