Evolving proteins in mammalian cells using somatic hypermutation.

Evolving proteins in mammalian cells using somatic hypermutation. Research Abstract Details 

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Evolving proteins in mammalian cells using somatic hypermutation. Abstract Text:

Lei Wang,Roger Y Tsien,Lei Wang,Roger Y Tsien,

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 approximately 1 d to mutate the target gene, approximately 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.

Evolving proteins in mammalian cells using somatic hypermutation. Publishing Authors By Initials

L Wang,RY Tsien,L Wang,RY Tsien,

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Evolving proteins in mammalian cells using somatic hypermutation. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: Nature protocols

VOLUME: 1

Page Numbers: 1346-50

Journal Abbreviation:

ISSN: 1750-2799

DAY: 3

MONTH: 12

YEAR: 2006

Evolving proteins in mammalian cells using somatic hypermutation. Information

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LANGUAGE: eng

NlmUniqueID: 101284307

Evolving proteins in mammalian cells using somatic hypermutation. Keywords Mesh Terms:

KEYWORDS: Proteins

MESH TERMS: genetics

Chemical & Substance for Abstract: Evolving proteins in mammalian cells using somatic hypermutation. Information

Substance Name: red fluorescent protein

Registry Number: 0

Grant and Affiliation Information for Evolving proteins in mammalian cells using somatic hypermutation.

AFFILIATION: The Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California 92037, USA. lwang@salk.edu

Country: England

AGENCY: United States NINDS

GRANT: NS27177

ACRONYM: NS

MEDLINETA: Nat Protoc

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