Fluorescent protein engineering by in vivo site‐directed mutagenesis

Abstract

Summary In vivo site-directed mutagenesis by ssDNA recombineering is a facile method to change the color of fluorescent proteins without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the E. coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the fluorescent protein. Eight different fluorescent proteins (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided.

Herpes ICP8 protein stimulates homologous recombination in human cells

“…Recombination was significantly improved as oligo length increased (by Fisher's Exact Test) in pairwise comparisons of each DNA length for a given oligo sequence in each cell line, suggesting that 65 nt oligos are better recombineering substrates in human cells than are shorter oligos. Although in our previous optimization studies in E. coli, little or no additional benefit was added by lengthening oligos beyond 70 nt (Valledor et al 2012), it remains to be determined if longer oligos could be more efficient for ICP8-stimulated recombineering.…”

“…proof of concept, we created a quantitative and sensitive reporter for genome editing using ssDNA oligos. We choose to change the emission spectrum of eGFP by targeting codon 203 (threonine, T) to code for another amino acid (tyrosine, Y) to yield a red-shifted fluorescent protein (referred to as Mostaza in (Valledor et al 2012) and Yellow in this report) (Figs. 2A and 2B).…”

“…2A and 2B). Lentiviral vectors based on pDual-eGFP (Valledor et al 2012) were created to transduce human 293T cells with lentiviruses carrying eGFP or eGFP T203Y . Green (eGFP) and Yellow (eGFP T203Y ) expressing cells could be distinguished and quantified by flow cytometry (Fig.…”

“…We called this synthetic protein "HumBeta". In this way, we attempted to mimic an efficient bacterial recombineering protocol (Valledor 2012) where the synaptases are expressed from stable transgenes in a burst of induction just before adding the oligo substrate for recombination. By comparing recombineering efficiency using a synaptase from a virus that co-evolved with humans and a synaptase that coevolved with a bacterium, we surmised we might probe host specificity of recombineering.…”

Single molecule techniques in DNA repair: A primer

“…They are large compared to organic fluorophores, such as fluorescein, at ~25 kDa versus 1 kDa in size [112]. However, unlike organic dyes, FPs can be genetically encoded to a protein of interest thus providing 100% labeling in a wide possible spectrum of colors [113]. This not only permits imaging of single proteins within cells, but also enables multicolor labeling of different single molecules, aiding tracking and analysis of protein movements and interactions [114].…”