Thursday, February 4, 2010

Recombinant Protein Expression: Part 1

Recombinant protein expression projects typically start with cloning the desired gene into appropriate expression vectors. These vectors can be specifically suited for bacterial, yeast, viral or mammalian expression. Bacterial versus mammalian expression is discussed here. For most protein expression needs, bacterial systems provide good yields, are easy to grow, maintain and expand and do not require expensive growth media.

Protein expression can either be constitutive (bacterial cells expressing desired protein continuously as they grow) or can be induced at a later stage in culture when the culture biomass is high but has not grown beyond the logarithmic phase of growth (uses active repression of expression).This is a specifically useful approach if the protein being expressed is toxic to the bacterial cells or if protein expression levels need to be modulated and fine-tuned. It becomes important in cases where the recombinant protein expression is very high and the protein tends to misfold and form inclusion bodies. There are multiple commercial bacterial expression systems available for inducible expression. Many new bacterial strains have also been developed that help in maintaining protein expression and solubility. Scale up is fast and the recombinant protein can either be secreted into the medium or harvested by bacterial cell lysis.

A major drawback of bacterial recombinant protein expression is the lack of appropriate protein modifications including post-translational processing like glycosylation. This is especially important when expressing a protein whose activity/ antigenicity is strongly dependent upon glycosylation. A lot of protein antigens and antibodies fall into this category. Mammalian expression addresses some of these issues very well although glycosylation patterns may vary between different cell lines chosen for expression. The most commonly used cell lines being HEK293T, CHO and NS0 cells. 293 cells are typically used for transient expression and CHO cells are used for the generation of cell lines stably expressing desired protein.

Part 2 will explore some of the current bottlenecks in mammalian protein expression.

"Is CHO based recombinant protein expression overhyped?" Comments welcome.

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