The Macromolecular Therapeutics Development Facility (MTDF) offers expertise in miniaturized and large-scale culturing in a range of expression platforms, including bacterial, mammalian (CHO or HEK293) and insect (Sf9 and High Five) systems.
The facility has the capability to perform high-throughput cloning and protein production screening at both small and large scales. Through automation and high-density culturing techniques the facility can screen hundreds of constructs in parallel for expression, and have the capability for scale-up production and purification of 48 targets in parallel. For high-throughput cloning, ligation independent cloning in a multiwall plate format utilizes a Beckman FxP liquid handling robot; and colony screening in multiwall blocks, automated using a custom built automated colony picking system. High-throughput cell lysis, in either multiwall format or 50ml tubes, is performed using a custom built EPSON Scara-based Sonication robot. Large scale parallel E. coli culturing is performed using a LEX 48 airlift fermenter. The expertise and equipment offered by the facility allow rapid screening of different vectors for identification of superior expression constructs, high-throughput site-directed mutagenesis with screening of mutated constructs for their effect on protein expression, and parallel expression analysis of panels of targets.
All of the cloning and mutagenesis services that the facility offers are also used to generate constructs for eukaryotic protein expression analysis, and production. Small-scale screening of protein production is offered at culture volumes as low as 1ml. The evaluation of protein production at small-scale has a two-fold benefit, in cost reduction and in allowing parallel screening of expression conditions, vectors and constructs. The facility has available a suite of 18 vectors from DNA 2.0, which allows screening of various elements of the construct (including promoter, terminator and mammalian replication origins), and determining how they effect protein production levels in transient expression systems. Small-scale screening can be carried out through transient transfections, lentiviral transduction or baculovirus infection. In order to facilitate high-throughput screening, we have a custom cell::explorer cell culture system, contained within a BSL-2 enclosure. Utilizing this system, it is possible to generate thousands of transfectants or viral particles in parallel.
The large-scale protein production capabilities offered by the facility include the growth of suspension cell-lines in optimized 5L culture vessels, with a nominal growth volume of 3L. These culture vessels provide significant enhancements compared to traditional shake flasks, and have been used to produce multi-milligram quantities of hundreds of secreted, glycosylated proteins. Additionally, for larger-scale production, the facility has two types of bioreactor; 25L capacity WAVE bioreactors and a pair of New Brunswick CelliGen 10.5L autoclavable bioreactors, which allow continuous monitoring, and can be used for batch, fed batch or continuous culture. These systems have allowed for the very large-scale production of growth factors (gram quantities).
In addition to our protein production capabilities, protein purification services at all scales, are offered, from small-scale screening of multiple samples to purification of protein from multi-liter production runs. The large-scale protein purification services extend to production of low endotoxin protein preparations (< 1 EU/mg), suitable for animal injection.
The MTDF is available for consultation, including contribution to grant applications; in addition to offering expertise at the planning stages of a project, we can enable PIs to submit grant applications can that become possible by utilizing the services, equipment and instrumentation that are offered.