Innate Immune Activation by Dominic De Nardo & Christine M. De Nardo

Author:Dominic De Nardo & Christine M. De Nardo
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

1.4 Limitations of LUMIER

The use of tagged proteins largely restricts LUMIER to well-transfectable cells, such as HEK293T cells. Second, Protein A and Renilla luciferase are relatively large fusion tags (137 and 310 amino acid residues, respectively), which could potentially influence the binding characteristics of proteins in which orientation (i.e. fusion to either amino- or carboxy terminus) often plays a crucial role. Thus, it is advisable to assess each tag at both termini and also swap Renilla and affinity tag between bait and prey. The potentially cumbersome generation of such constructs can be greatly improved if restriction enzyme-independent cloning methods are employed to generate the necessary fusion constructs. We recommend setting up a plasmid library of commonly studied proteins with constructs for all possible combinations of tags. The Gateway system (originally Invitrogen) in our hands has proven as a powerful tool for fast and efficient cloning. In this way, multiple tagged constructs can rapidly be generated and assayed. LUMIER thus allows for medium-throughput screening of entire pathways, or of multiple mutants or sequence variants against a given bait.

We have successfully used LUMIER to probe the effect of rare germline [5] and recurrent somatic oncogenic mutations in MYD88 [8]. However, the principles of LUMIER can be extended to other post-receptor complexes or signaling cascades. In the following, we describe the transfection of HEK293T cells with LUMIER-compatible expression constructs in a 96-well format (Subheading 3.1). Subheadings 3.2–3.4 illustrate cell harvest and luciferase measurement, whereas different models of data analysis are summarized in Subheading 3.5.

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