Engineering color variants of green fluorescent protein (GFP) for
thermostability, pH-sensitivity, and improved folding kinetics
Naser Aliye1,2, Attilio Fabbretti1, Giulio Lupidi3, Tsepo Tsekoa4 , and Roberto
Spurio1
SUPPLEMENTARY FIGURES
Figure S1. SDS gel electrophoresis of crude and purified GFP variants.
Lane 1, Precision Plus Protein™ Dual Color Standards (BIO-RAD); Lane 2, crude FF-GFP; Lane 3, heattreated FF-GFP; Lane 4, GFP (ASV); Lane 5, GFP30R; Lane 6, GFP39N; Lane 7, FF-GFP; Lane 8, AcSCFP; Lane 9, FFTS-YFP. Four microgram of pooled fractions of each GFP variant were applied on 12%
polyacrylamide gel.
Figure S2. The molar extinction coefficient (ε) of the GFP variants. The molar absorptivity (extinction)
coefficient was determined by measuring the absorption of increasing concentration of the proteins at
absorption maxima of the fluorophore in 1cm path-length quartz cuvette at 25°C using BioTek® PowerWave
HT Microplate Spectrophotometer (Bio-Tek Instruments). The graphs report the experimental values
obtained for GFP (ASV) parental material (A), GFP30R (B), GFP39N (C), FF-GFP (D), AcS-CFP (E) and
FFTS-YFP (F). The extinction coefficient was computed using Lambert-Beer Law. The data were computed
using linear regression analysis.
Figure S3. Determination of the acid dissociation constant (pKa) of the GFP variants. Following the
procedure described in the material and methods section, the experimental data were processed to obtain pKa
values. A, GFP (ASV) parental material; B, GFP30R; C, GFP39N; D, FF-GFP; E, AcS-CFP and F, FFTSYFP. The absorbance values are normalized at the maximum absorbance peak. Except for AcS-CFP (E), the
pKa values of all the GFP variants were determined by fitting the data of absorbance values recorded at the
excitation maxima at each pH. Note the behavior of the pH-unresponsive cyan protein variant (E).
SUPPLEMENTARY TABLES
Table S1. List of oligonucleotide primers used in the study.
Code
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
P16
P17
Primer sequence (5’-3’)
CAACACTTGTCACTACTTTGACTTATGGTGTTCAATGCTTTG
CAAAGCATTGAACACCATAAGTCAAAGTAGTGACAAGTGTTG
GCACAAATTTTCTGTCAGAGGAGAGGGTGAAGGTG
CACCTTCACCCTCTCCTCTGACAGAAAATTTGTGC
GTGAAGGTGATGCAACAAACGGAAAACTTACCCTTAA
TTAAGGGTAAGTTTTCCGTTTGTTGCATCACCTTCAC
GGAAAGAACTATATCTTTCAAAGATGACGGGACCTACAAGACACGTG
CACGTGTCTTGTAGGTCCCGTCATCTTTGAAAGATATAGTTCTTTCC
GAATGGAATCAAAGCTAACTTCAAAATTAGACACAACGTTGAAGATGGAAGCG
CGCTTCCATCTTCAACGTTGTGTCTAATTTTGAAGTTAGCTTTGATTCCATTC
CAACCATTACCTGTCCTATCAATCTGCCCTTTCG
CGAAAGGGCAGATTGATAGGACAGGTAATGGTTG
CAAATTGGAATACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAG
CTTTTGTTTGTCTGCCGTGATGTATACATTGTGTGAGTTAAAGTTGTATTCCAATTTG
CTTGTCACTACTTTCACTTGGGGTGTTCAATGCTTTG
CAAAGCATTGAACACCCCAAGTGAAAGTAGTGACAAG
ATTGATGAGCGGCCGCAACTGATGCAGCGTAGTTTTCGTC
F and R indicate forward and reverse primers used in DNA amplification reactions, respectively.
Remark
GFP F64L + G65T (F)
GFP F64L + G65T (R)
GFP S30R (F)
GFP S30R (R)
GFP Y39/N (F)
GFP Y39/N (R)
GFP F99/S + N105/T (F)
GFP F99/S + N105/T (R)
GFP V163/A + I171/V(F)
GFP V163/A + I171/V (R)
GFP T203Y to obtain yellow fluorescent protein (F)
GFP T203Y to obtain yellow fluorescent protein (R)
GFP Y145F and M153T (F)
GFP Y145F and M153T (R)
GFP Y66W to obtain cyan fluorescent protein (F)
GFP Y66W to obtain cyan fluorescent protein (R)
The reverse distal primer used in TPCR for multiple sitedirected mutagenesis in plasmid pGFP (ASV)
Table S2. The physicochemical characteristics of GFP variants.
a
GFP variant
pKa (Abs) a
Φb
ε (M-1.cm-1) c
Brightness d
GFP (ASV)
6.9 ± 0.14
0.44
38520
17.10
GFP30R
6.5 ± 0.16
0.58
47850
27.76
GFP39N
6.9 ± 0.16
0.59
49230
29.27
FF-GFP
6.9 ± 0.17
0.64
71750
46.06
AcS-CFP
pH-unresponsive e
0.35
36710
13.17
FFTS-YFP
7.4 ± 0.07
0.68
94810
64.88
Venus f
6.0
0.57
92200
52.5
SF-GFP g
nd
0.65
83300
54.1
EGFP h
5.4
0.67
55000
33.6
Emerald i
nr
0.68
57500
37.3
pKa values were determined based on absorbance at different pH at fixed absorbance maxima
wavelength as described in the Experimental Procedure section.
b The
fluorescence quantum yield (Φ) of the GFP variants were calculated by measuring the
integrated fluorescence intensity in TE buffer pH 8.0 (refractive index, η = 1.35) against quinine
sulphate in 0.1 M H2SO4 (refractive index, η = 1.33) used as a standard with quantum yield = 0.54
and applying Formula (I) described in the text.
c The
extinction coefficient were computed using Lambert-Beer Law.
d Brightness
e This
= (Φ*ε)/1000.
protein variant was found to be unresponsive to changing pH.
f
Venus GFP (Nagai et al. 2002);
g
Superfolder GFP (Pedelacq et al. 2006);
h
Enhanced GFP (Zhang et al. 1996)
i
Emerald GFP (Cubitt et al. 1999).
nd, not determined in this study. nr, not reported.