TY - JOUR
T1 - FACS-optimized mutants of the green fluorescent protein (GFP)
AU - Cormack, Brendan P.
AU - Valdivia, Raphael H.
AU - Falkow, Stanley
N1 - Funding Information:
We gratefullay cknowledgMe ark Troll for fluorimetric analysisH, arryNoller for use of his oligo synthesizearn d Rachel Green for help with oligo synthesisa nd for a carefurl eadingo f the manuscripwt;e thankG regVerdine for supplyingp KEN2 and Stan Tabor for pT7.7. This work was supportebdy a grantf romthe NIH (AI 36396), and by an unrestrictegdi ft from Praxis Pharmaceuticals to S.F.B.P.C. is supportedb y a Helen Hay Whitney postdoctorafel llowship.
PY - 1996
Y1 - 1996
N2 - We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FAGS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
AB - We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FAGS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
KW - FITC
KW - Fluorescence intensity
KW - Fluorescence-activated cell sorter
KW - GFP mutation
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U2 - 10.1016/0378-1119(95)00685-0
DO - 10.1016/0378-1119(95)00685-0
M3 - Article
C2 - 8707053
AN - SCOPUS:0029973636
SN - 0378-1119
VL - 173
SP - 33
EP - 38
JO - Gene
JF - Gene
IS - 1
ER -