Protein Engineering and Expression: Part 2; The Antibody Way

Mammalian Protein Expression bottlenecks will be discussed here in the context of antibody expression issues.

Past major issues:
•    Low or no antibody expression from the vectors being used
•    Longer selection time with Neomycin and more false positive clones with Zeocin or Puromycin selection
•    Positive FACS profiles for ‘LC only’ expression clones
•    No optimization of HC: LC ratio in transfections for stable expression
•    Transient expression results unmatched with stable cell line data

A majority of these can be attributed to:

1.    Promoter silencing and unstable integration

Examples of Problems reported by users:

Q1. I have cloned my gene of interest into mammalian expression vector pcDNA3.1. Transient transfection from this construct gives very good antibody expression but every time I select a stable pool, the cells that survive are resistant to the selection antibiotic but seems to have lost expression of my antibody genes? Why is that so?

Q2. I am selecting a cell pool after transfecting CHO-S cells with my antibody construct. After selection, the cells show no antibody expression even though they are still Zeocin resistant. I did see cells dying when I started selection and the resistant pool emerged 8- 10 days after selection…This has happened multiple times only for this ‘hard to express’ antibody.

A. The selection pressure for cell survival ensures that the antibiotic selection marker gene is maintained in the cells and is not silenced but the gene of interest is either actively silenced or excised out since it affords no survival advantage for the cell. Problems such as these have been solved using chromatin opening elements or using more stringent selection such as cloning the desired gene under a strong promoter followed by the selection marker gene placed downstream of an IRES element. Expression of both the desired gene and the selection marker from the same transcript ensures that antibody expression is not silenced. This will be discussed in detail in future posts.

Random integration events : Will be discussed in the next post.

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.