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.