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Evolving proteins in mammalian cells using somatic hypermutation


We describe a new method to mutate target genes through somatic hypermutation (SHM) and to evolve proteins directly in living mammalian cells. Target genes are expressed under the control of an inducible promoter in a B-cell line that hypermutates its immunoglobulin (Ig) V genes constitutively. Mutations can be introduced into the target gene through SHM upon transcription. Mutant genes are then expressed and selected or screened for desired properties in cells. Identified cells are subjected to another round of mutation and selection or screening. This process can be iterated easily for numerous rounds, and multiple reinforcing mutations can be accumulated to produce desirable phenotypes. This approach bypasses labor-intensive in vitro mutagenesis and samples a large protein sequence space. In this protocol a monomeric red fluorescent protein (mRFP1.2) was evolved in Ramos cells to afford a mutant (mPlum) with far-red emission. This method can be adapted to evolve other eukaryotic proteins and to be used in other cells able to perform SHM. For each round of evolution, it takes 1 d to mutate the target gene, 0.5–1 d to select or screen, and 2–4 d to propagate the cells for the next round depending on how many cells are collected.

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Figure 3: Setup for fluorescence ratio sorting using the BD FACSVantage flow cytometer.


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This work was supported by the Howard Hughes Medical Institute, National Institutes of Health Grant NS27177, and US Department of Energy Grant DE-FG03-01ER63276 to R.Y.T. L.W. was supported by the Damon Runyon Cancer Research Foundation (Merck Fellow, Grant DRG-1767-03), the Searle Scholars Program, and the Beckman Young Investigators Program.

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Wang, L., Tsien, R. Evolving proteins in mammalian cells using somatic hypermutation. Nat Protoc 1, 1346–1350 (2006).

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