Optimization of mNeonGreen for Homo sapiens increases its fluorescent intensity in mammalian cells

Abstract

Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.

Fig 1. DNA sequence and plasmid maps of mNeonGreen.

(A) Comparison of the DNA sequence of humanized mNeonGreen with that of the original one. A pairwise alignment of two DNA sequences of humanized (GenBank Accession No. LC279210) and original mNeonGreen (GenBank Accession No. KC295282) was performed using a CLUSTAL W program (http://clustalw.ddbj.nig.ac.jp/). (B) Plasmid maps for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG. Humanized mNeonGreen cDNA with a triple Gly-Gly-Gly-Ser linker was inserted into the NheI-BglII site of pAcGFP-C1 after removing AcGFP cDNA to create pmNeonGreenHO-G. Humanized mNeonGreen-3xFLAG cDNA was inserted into the NheI-BglII site of pAcGFP-C1 after removing AcGFP cDNA to create pmNeonGreenHO-3xFLAG. MCS, multicloning sites; CMV, cytomegalovirus.

Fig 2. Original and humanized mNeonGreen fluorescence in HEK293.

Plasmids designed for expression of original or humanized mNeonGreen were transfected into HEK293 cells. As a control of transfection efficiency, mCherry2-C1 was employed. Fluorescent intensity of mNeonGreen and mCherry2 was obtained 48 h after transfection using a 2300 EnSpire multimode reader. The data were analyzed with a Welch’s t-test; p < 0.01 in green fluorescent intensity of mNeonGreen; p > 0.7 in red fluorescent intensity of mCherry2. Graphs show the relative fluorescent intensity of both fluorescent proteins (%).

Fig 3. Mitochondrial distribution of humanized mNeonGreen tagged with a mitochondria-targeting signal.

The plasmid pmNeonGreen-mito was transfected into COS1 cells. 48 h after transfection, the green fluorescent images in the cells were monitored using a BZ-X700 microscope. Bar indicates 50 μm.

Fig 4. Expression of humanized mNeonGreen-3xFLAG in HEK293 cells.

A pmNeonGreenHO-3xFLAG plasmid for expression of humanized mNeonGreen-3xFLAG was introduced into HEK293 cells. Cells were lysed 24 h after transfection, and total proteins in the lysate were separated on 12.5% SDS-polyacrylamide gels. The mNeonGreen-3xFLAG fusion protein was detected with an anti-FLAG M2 antibody. As a loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was recognized using anti-glyceraldehyde-3-phosphate dehydrogenase antibody.