protein_expression_purThe CBSE expression facilities allow for large scale cloning, expression, purification, solubility, and refolding studies. Structural  studies on the different classes of proteins depend highly on the development of suitable expression systems. Various vectors and host cell expression systems have been engineered, spanning from simple prokaryotic vectors to complicated eukaryotic expression methods.

Our experience includes working with:

Prokaryotic Expression Systems

Bacterial expression systems have been used extensively, especially those based on E. coli. We use a variety of vectors to control expression levels by use of different promoters, induction systems, and the E. coli expression hosts. These vectors can also place a tag on the protein of interest to increase solubility or assist in purification.

Eukaryotic Expression Systems
Baculovirus is utilized should proteins require glycosylation for proper expression and folding. Several insect cell lines are used to optimize expression.

protein_expression_pur_2Additional eukaryotic expression capabilities include the combination of lentiviral vectors with a variety of human cell lines. LV systems have shown higher biological activity levels for human enzymes implying that the protein conformation/folding is more representative of the in-vivo situation (an important consideration when designing drugs/peptides based on the structural data). We have developed and exploited lentiviral vectors (LVV) for the rapid generation of mammalian cell lines stably expressing high-levels of recombinant proteins. Inducible expression systems can be used to overcome expression problems associated with toxicity. These systems have been highly effective with industrial partners interested in the production of difficult to express proteins such as membrane and cytotoxic proteins.

The key assumption underlying the design of our purification strategy is that the initial quantities of protein available will be small, even with excellent expression levels. Therefore, we can quickly begin performing preliminary testing, protocol development, and characterization with a relatively small starting sample—typically 2-3 mg of crude target protein. Candidate proteins that are identified as promising during this preliminary work will be flagged for subsequent high-level expression and scaled-up purification. This strategy ensures that the most amenable proteins are identified as quickly as possible. As we aim to implement efficient, high throughput methods for protein purification, we often employ microscale chromatography and novel biophysical characterization methods to rapidly and efficiently optimize purification parameters for each target protein. The material is characterized in terms of purity, folding, and aggregation state. For proteins that show acceptable behavior at this stage, large-scale expression and scaled-up purification efforts are initiated in support of extensive crystallization trials. The CBSE has developed novel approaches for rapid determination of protein specific solution conditions that reduce or eliminate unwanted aggregation and promote stability. Our unique approach has been validated with both aqueous and membrane proteins providing critical information for pharmaceutical companies with an interest in designing protein therapeutics with optimal solution properties (particularly for vaccines).