Protein Purification

Overview

The Protein Purification Facility (PPF) is equipped to support research groups at the London Research Institute with queries regarding expression and purification of recombinant proteins. The PPF is closely associated with the LRI research groups employing x-ray crystallography to determine three-dimensional protein structures in order to understand the molecular basis for protein function and/or dysfunction in disease states.

Additionally, PPF collaborates with research groups with a need for high quality protein for other downstream applications, such as enzymatic assays.

Depending on the nature of the protein/project, PPF offers three expression technologies:

• E. coli based expression
  Generally, the first choice for protein expression due to it being a well-established technology with a wide range of differently tagged vectors available and its relative low cost of implementation.
  
  
• Baculovirus mediated expression in insect cells
  The baculovirus (BV) technology is the central expression technology employed at the PPF. It offers good yields and post-translational modifications of both intra- as well as extracellular proteins. Hence, it is often used to produce proteins, that can not be expressed/modified satisfactorily in E .coli. It generally takes 2-3 weeks to generate high-titer virus to infect either Sf9 or Hi-5 insect cells.
  
  
• Mammalian cell-based expression
  Secreted proteins are, in certain cases, most efficiently produced in mammalian cells. Once a stable cell-line, secreting the protein of interest, is generated, it can be expanded to grow in roller flasks and continuously produce the desired protein without the need to repeatedly infect with baculovirus. Typically used cell-lines are CHO and HEK293.

Most recombinant proteins contain cleavable affinity tags to facilitate purification using either immobilised metal affinity chromatography or commonly used tags such as GST or MBP. Additionally, the PPF uses the ÄKTA™ Explorer platform for conventional chromatography such as ion-exchange, hydrophobic interaction and size exclusion chromatography.