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

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.

To utilize mNeonGreen in mammalian cells more effectively, it is important to obtain efficient expression. Previously, mNeonGreen cDNA was found to contain at least seven codons used rarely in H. sapiens [3]. Therefore, we hypothesized that expression of mNeonGreen in mammalian cells would improve upon optimization of its codons for H. sapiens. Another disadvantage of mNeonGreen versus mEGFP and EGFP is that there are few antibodies available for mNeonGreen recognition. While there are many commercially available antibodies for GFP and its other variants, they cannot be used to detect mNeonGreen due to the low amino acid sequence homology between mNeonGreen and mEGFP (Table 1). Therefore, we also generated a plasmid that would help optimize expression of 3xFLAG-tagged, humanized mNeon-Green [11]. These improvements will be very useful for analyzing biological events using this protein in mammalian cells.

DNA synthesis and construction of plasmids for expression of humanized mNeonGreen and mNeonGreen-3xFLAG
mNeonGreen amino acid sequence (GenBank Accession No. AGG56535.1) codon optimization for H. sapiens was performed using MacVector software based on the codon usage database on the Kazusa DNA Research Institute website (http://www.kazusa.or.jp/codon/cgi-bin/ showcodon.cgi?species=9606). The resultant optimized DNA fragment was synthesized by Integrated DNA Technologies with a linker sequence (5'-AAAAAAGCTAGCGCCACC-3') before the start codon and another (5'-AAGTCCGGAACTAGTTTTTT-3') before the stop codon. The synthesized DNA fragment was digested by restriction enzymes NheI and BspEI, and a NheI-BspEI site was introduced to pAcGFP-G; pAcGFP-G is similar to pAcGFP-C1 and contains a triple Gly-Gly-Gly-Ser linker sequence at the C-terminus before its multicloning site. The resultant plasmid was designated as pmNeonGreenHO-G (Fig 1). For the expression of original mNeonGreen, a DNA fragment of mNeonGreen (GenBank Accession No. AGG56535.1) was synthesized by Integrated DNA Technologies with a linker sequence (5'-AAAAAAGCTAGCGCCACC-3') before the start codon and another (5'-AAGTCCGGAA CTAGTTTTTT-3') before the stop codon. The plasmid used for expression of original mNeonGreen (pmNeonGreen-G) was constructed using the same strategy as for humanized mNeonGreen above.
For the expression of mitochondria-localized humanized mNeonGreen protein, we amplified a DNA fragment encoding a mitochondrial targeting signal of ActA gene of Listeria Table 1. Comparison of the amino acid sequence of mNeonGreen with that of EGFP using Clustal-W alignment.

Fluorescence microscopy
Cells were fixed in fixation solution (4% paraformaldehyde in phosphate-buffered saline) at room temperature for 5 min, and fluorescence was monitored using a BZ-X700 microscope (Keyence, Tokyo, Japan).

Analyses of fluorescence intensity of humanized mNeonGreen
The plasmids, pmNeonGreen-G or pmNeonGreenHO-G was transfected into HEK293 cells. As a control of transfection efficiency, mCherry2-C1 was employed. At 48 h after transfection, the fluorescent intensity of mNeonGreen and mCherry in 293 cells was analyzed using a 2300 EnSpire multimode reader (PerkinElmer, Massachusettes).

Increased fluorescent intensity of humanized mNeonGreen in mammalian cells
mNeonGreen is one of brightest GFPs derived from Branchiostoma lanceolatum. To investigate whether the reported cDNA sequence of mNeonGreen (GenBank Accession No. KC295282) is suitable for expression in human cells, we analyzed its codon usage with the Kazusa DNA Research Institute website (http://www.kazusa.or.jp/codon/cgi-bin/showcodon. cgi?species=9606) [ Table 2].
The original mNeonGreen mRNA sequence was found to contain some codons that are rarely used in H. sapiens, especially two UUA (7.7 frequency per 1000 codons), three UCG (4.4 frequency per 1000 codon), one CCG (6.9 frequency per 1000 codons) and one CGU (4.5 frequency per 1000 codons) [ Table 3]. Therefore, we concluded that optimization of the mNeon-Green codon sequence for H. sapiens would improve its expression and thereby, its fluorescent intensity, in mammalian cells.
Accordingly, we changed 51 out of 236 codons within mNeonGreen (GenBank Accession No. KC295282) to more frequently used codons in H. sapiens, synthesized a DNA fragment encoding humanized mNeonGreen (GenBank Accession No. LC279210), and generated our expression pmNeonGreenHO-G (Fig 1). The resultant plasmid was transfected into HEK293 cells, and the fluorescent intensity of mNeonGreen in the cells 24 h after transfection was examined. As a control for transfection efficiency, mCherry2-C1 was employed. The fluorescent intensity of humanized mNeonGreen was significantly increased (1.39 ± 0.06-fold, p<0.01) compared with that of original mNeonGreen, while there is no significant difference of that of mCherry2 (Fig 2). This increase was obtained by substituting codons within original mNeon-Green cDNA that are rarely used in humans to more highly used codons. Especially, the codons, UUA, UCG, CGU, and CCG, are rarely used in Mus musculus and Rattus rattus, therefore the fluorescent intensity of humanized mNeonGreen will be increased in mouse and rat cell lines.

Humanized mNeonGreen with a mitochondria-targeting signal was showed mitochondria-distribution
To investigate whether the pmNeonGreenHO-G is suitable for monitoring intracellular organelle, we generated a plasmid, pmNeonGreenHO-mito, for the expression of a mNeonGreen with a mitochondria-targeting signal. This plasmid was transfected into COS1 cells, and its green fluorescence in the cells were investigated. As shown in Fig 3, the humanized mNeonGreen with a mitochondria-targeting signal showed a representative distribution of mitochondria. Therefore, at least, the pmNeonGreenHO-G is available to monitor mitochondria morphology. The original mNeonGreen is available to investigate the other organelle-morphology [3]. Considering the codon-optimization for Homo sapiens leads to no change of amino acid sequence of mNeonGreen protein itself, this plasmid can be used for monitoring the intracellular distribution of a certain protein as a fusion protein

Generation of mNeonGreen-3xFLAG expression plasmid
Clustal-W analyses revealed the amino acid sequence of mNeonGreen has low homology with that of EGFP (27% identity, 14% similarity; Table 1), indicating most commercially available anti-GFP antibodies will not react with mNeonGreen. To solve this problem, we generated the pmNeonGreenHO-3xFLAG expression plasmid (Fig 1). The plasmid was introduced into HEK293 cells and lysates prepared 24 h after transfection. Total proteins were separated by SDS-polyacrylamide gel electrophoresis, and the mNeonGreen-3xFLAG protein was recognized with an anti-FLAG-M2 antibody (Fig 4). As expected, an approximately 33 kDa band  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.
https://doi.org/10.1371/journal.pone.0191108.g004 corresponding to mNeonGreen-3xFLAG was recognized. FLAG tags are one of the most employed epitopes for biochemical and cellular biology analyses. In conclusion, our pmNeon-GreenHO-3xFLAG plasmid and mNeonGreen-3xFLAG fusion protein system will provide certain advantages to biochemical studies in mammalian cells. The lysate of cells transfected into pmNeonGreenHO-3xFLAG (mNeonGreenHO-3xFlag) or a control plasmid (CON) was separated on SDS-PAGE, and Flag-tagged proteins and GAPDH were recognized. From the left, molecular weight marker (Mk, lanes 1 and 7), negative control (CON) (lanes 2 and 8), mNeonGreen-3xFlag (lanes 3-6 and 9-12). (B) The original data of Humanized mNeonGreen with 3xFlag tag using a rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. As a loading control, GAPDH in the lysate was recognized after removing anti-Flag-M2 antibody. (TIFF)