Generation of Destabilized Green Fluorescent Protein as a Transcription Reporter

The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422–461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422–461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFκB binding sequences and monitoring tumor necrosis factor α-mediated NFκB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFκB translocation to the nucleus. The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422–461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422–461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFκB binding sequences and monitoring tumor necrosis factor α-mediated NFκB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFκB translocation to the nucleus. Because of its easily detected green fluorescence, the green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; MODC, mouse ornithine decarboxylase; dEGFP, destabilized EGFP (EGFP-MODC-(422–461) fusion); secreted alkaline tumor necrosis 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; MODC, mouse ornithine decarboxylase; dEGFP, destabilized EGFP (EGFP-MODC-(422–461) fusion); secreted alkaline tumor necrosis the is a widely used reporter in studies of gene expression and protein localization GFP fluorescence not require it is to use it in for detection The fluorescence of is the key sequence acids This sequence to a GFP of to amino and to and is by a gene with of GFP and the that the GFP a GFP is a of amino acids of amino acids that an to the the is the it the The GFP stable a of with The stability of GFP limits its application in induction widely in in is by that protein the is a sequence such as the PEST a sequence correlated with protein degradation the is a such as a to is by a of and by a not require for such protein is mouse ornithine decarboxylase (MODC) is the key in the of This protein is to be of the in and its half-life is ornithine decarboxylase of is stable when in of the sequences of sequences its that not for ornithine decarboxylase This a PEST and its its rapid degradation the C-terminal is also for degradation of ornithine decarboxylase in unstable fusion with the this as the of that PEST sequences correlate with protein degradation is a a with and This is by amino The of the PEST sequence in protein degradation in several such as that the of mouse ornithine this the degradation domain of mouse ornithine decarboxylase to the of The degradation domain of the half-life of EGFP in to 2 h. We mutated key amino acids in the PEST sequence of the fusion protein and identified several mutants with half-lives. of the fusion protein to the binding sequences of the transcription factor NFκB detection of NFκB induction in cells. The use of destabilized EGFP as a transcription reporter gene induction in time with cells. The of induction with the biochemical such as translocation was also EGFP and the of with EGFP was with that sequence the end and sequence the The of EGFP was to a with the of The of was with that a sequence the end and an sequence the The and the fusion was the expression for use in the expression this EGFP-MODC-(422–461) and its amino acids of the PEST sequence in the fusion protein mutated to The mutants a The and with and transfected for degradation studies. a fusion protein of the and the and can be used for expression of the transcription by binding to a with binding in the This binding can be by and the expression can be by the of in the transfected to as the fluorescence of EGFP the the for and with for The a and a fluorescence protein the with cycloheximide a of for transfected with by and the in of The for fluorescence a EGFP was and was detected a transfected with in and by by a and EGFP and fusion detected a GFP The detection was with the Western detection a of NFκB binding sequence the with the The gene was with the EGFP and transfected cells. the with The the for NFκB translocation and induction of dEGFP, transfected with the transfected with in for and with The with a of in for h. The with NFκB in for 2 h. with the with for in with the in of MODC, amino acids ornithine decarboxylase degradation in This a PEST sequence amino acids the PEST domain can also EGFP it to the C-terminal end of EGFP to This fusion was a expression The fluorescence of the fusion protein was a fluorescence it was in cells. that the fluorescence of EGFP-MODC-(422–461) is similar to EGFP with in the tested the of the of to EGFP degradation in The was transfected cells. the with for and and the in fluorescence of the transfected was by fluorescence The fluorescence of the fusion protein in the as was that the fusion protein is a with fluorescence had by The that the half-life of the fusion protein is h. did not a in the fluorescence in the the half-life of the fusion protein used to the in fluorescence of the fusion protein. of the fluorescence a that the half-life of the fusion protein is 2 h. had of fluorescence to the analysis of the fluorescence of EGFP and transfected with the used in the transfected with for and h. The with and to the of the fluorescent was the the half-life of the EGFP fusion protein and its fluorescence, the EGFP and EGFP-MODC-(422–461) transfected used for also used for Western blot analysis with a in the was to EGFP and the fusion protein. in the of EGFP was However, the EGFP-MODC-(422–461) fusion protein was as by a of the fusion protein was 2 of We that the half-life of the EGFP-MODC-(422–461) fusion protein is h. that the fluorescence decay of the fusion protein with its protein The fusion protein was blot analysis of protein of EGFP and used for The to and a EGFP and the EGFP fusion protein detected with a the of PEST amino acids to protein degradation in dEGFP, mutated and of dEGFP, as as the amino to degradation by the in fluorescence The for fusion mutants in We that the degradation was not to the of PEST was by such as the of the amino the protein, of the amino acids and the of the 2 that the and of the PEST sequence to protein in of the PEST sequence of EGFP-MODC-(422–461) and the of the analysis of EGFP, and in transfected was in the as with for and and for fluorescence by The fluorescent time as a of in a can be used as a transcription it to of NFκB binding sequence and the The was transfected for monitoring NFκB The time course induction with kinetics is similar to used reporter. of and 2 was and The that can be used as a reporter to transcription unmodified EGFP, when used as the reporter for did not to the This result be of a of the stable protein in the cells. unlike dEGFP, a rapid stable EGFP be used as a reporter for the of NFκB transfected by the reporter the with The for analysis with of fluorescence was EGFP, is an protein not require a to use it to the transcription induction and NFκB translocation the to the nucleus. with with for and and with NFκB and with a in induction was with and the translocation of NFκB with NFκB was in the of rapid translocation of NFκB the to the nucleus. This was was not detected this 2 NFκB had in the and the induction of NFκB the to the and the induction of was h. The that the translocation of NFκB is in response to and that of the NFκB was to the h. This translocation of NFκB in its in the of transcription induction of detection of NFκB localization and induction of transfected by the with for and h. NFκB translocation was with and induction was by NFκB translocation and induction of detected destabilized EGFP by the degradation domain of to the of The fusion protein is unstable in the presence of with indicated that the half-life of is 2 h. Western blot analysis indicated that the half-life was h. The in the half-life by of the presence of and of the EGFP is and with a of GFP, Western and the result in half-life Because to use GFP as a reporter the fluorescence the protein its fluorescence half-life is a We that the half-life of is 2 of that EGFP degradation in a PEST sequence amino acids to amino and and analysis and is for protein However, that degradation not correlate with the of PEST mutants such as This in a with in the of is correlated to the of in the sequence the PEST by the of PEST sequences of to protein Because it is easily EGFP is an reporter for studies of protein rapid of the rapid of allows its application studies destabilized GFP, such as studies in the its rapid in accumulation in to in the when that a stable this fusion in of unmodified EGFP be the destabilized of EGFP can be used as a reporter in the of the of as a transcription reporter to that of NFκB activation. The kinetics of time course induction of and dose response similar to of the kinetics of EGFP The for the is that of its EGFP is easily the of the induction of EGFP is that of the rapid of its accumulation in time course induction similar to was also to the induction of by not that is of as a transcription reporter. Because fluorescence can be detected in time the of it an in the such as that reporter with can be used for in and to of induction can be directly by monitoring the of the fluorescence with a of fluorescence of destabilized GFP by the to transcription transcription factor NFκB expression of a of to and response of NFκB rapid degradation of its and translocation of the NFκB the to the degradation of is a rapid The binding of NFκB can be detected degradation The degradation of is and it of the The of is also that it protein Because is the gene of it is that the of is by The not the binding of NFκB a for NFκB to induction of the that of and the NFκB is this that the of NFκB in the is also the of NFκB protein to h. Because was used as a reporter in this induction can be with the of We that the of NFκB in the is with its induction of the reporter gene use of the protein as a transcription reporter allows monitoring of transcription induction directly and its with biochemical Because of its easily detected green fluorescence, the green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; MODC, mouse ornithine decarboxylase; dEGFP, destabilized EGFP (EGFP-MODC-(422–461) fusion); secreted alkaline tumor necrosis 1The abbreviations used are: GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; MODC, mouse ornithine decarboxylase; dEGFP, destabilized EGFP (EGFP-MODC-(422–461) fusion); secreted alkaline tumor necrosis the is a widely used reporter in studies of gene expression and protein localization GFP fluorescence not require it is to use it in for detection The fluorescence of is the key sequence acids This sequence to a GFP of to amino and to and is by a gene with of GFP and the that the GFP a GFP is a of amino acids of amino acids that an to the the is the it the The GFP stable a of with The stability of GFP limits its application in induction studies. widely in in is by that protein the is a sequence such as the PEST a sequence correlated with protein degradation the is a such as a to is by a of and by a not require for such protein is mouse ornithine decarboxylase (MODC) is the key in the of This protein is to be of the in and its half-life is ornithine decarboxylase of is stable when in of the sequences of sequences its that not for ornithine decarboxylase This a PEST and its its rapid degradation the C-terminal is also for degradation of ornithine decarboxylase in unstable fusion with the this as the of The that PEST sequences correlate with protein degradation is a a with and This is by amino The of the PEST sequence in protein degradation in several such as that the of mouse ornithine this the degradation domain of mouse ornithine decarboxylase to the of The degradation domain of the half-life of EGFP in to 2 h. We mutated key amino acids in the PEST sequence of the fusion protein and identified several mutants with half-lives. of the fusion protein to the binding sequences of the transcription factor NFκB detection of NFκB induction in cells. The use of destabilized EGFP as a transcription reporter gene induction in time with cells. The of induction with the biochemical such as translocation was also EGFP and the of with EGFP was with that sequence the end and sequence the The of EGFP was to a with the of The of was with that a sequence the end and an sequence the The and the fusion was the expression for use in the expression this EGFP-MODC-(422–461) and its amino acids of the PEST sequence in the fusion protein mutated to The mutants a The and with and transfected for degradation studies. a fusion protein of the and the and can be used for expression of the transcription by binding to a with binding in the This binding can be by and the expression can be by the of in the transfected to as the fluorescence of EGFP the the for and with for The a and a fluorescence protein the with cycloheximide a of for transfected with by and the in of The for fluorescence a EGFP was and was detected a transfected with in and by by a and EGFP and fusion detected a GFP The detection was with the Western detection a of NFκB binding sequence the with the The gene was with the EGFP and transfected cells. the with The the for NFκB translocation and induction of dEGFP, transfected with the transfected with in for and with The with a of in for h. The with NFκB in for 2 h. with the with for in with the in The EGFP and the of with EGFP was with that sequence the end and sequence the The of EGFP was to a with the of The of was with that a sequence the end and an sequence the The and the fusion was the expression for use in the expression this EGFP-MODC-(422–461) and its amino acids of the PEST sequence in the fusion protein mutated to The mutants a The and The with and transfected for degradation studies. a fusion protein of the and the and can be used for expression of the transcription by binding to a with binding in the This binding can be by and the expression can be by the of in the transfected to as the fluorescence of EGFP the the for and with for The a and a fluorescence protein the with cycloheximide a of for The transfected with by and the in of The for fluorescence a EGFP was and was detected a The transfected with in and by by a and EGFP and fusion detected a GFP The detection was with the Western detection a of NFκB binding sequence the with the The gene was with the EGFP and transfected cells. the with The the for NFκB translocation and induction of dEGFP, transfected with the transfected with in for and with The with a of in for h. The with NFκB in for 2 h. with the with for in with the in of MODC, amino acids ornithine decarboxylase degradation in This a PEST sequence amino acids the PEST domain can also EGFP it to the C-terminal end of EGFP to This fusion was a expression The fluorescence of the fusion protein was a fluorescence it was in cells. that the fluorescence of EGFP-MODC-(422–461) is similar to EGFP with in the tested the of the of to EGFP degradation in The was transfected cells. the with for and and the in fluorescence of the transfected was by fluorescence The fluorescence of the fusion protein in the as was that the fusion protein is a with fluorescence had by The that the half-life of the fusion protein is h. did not a in the fluorescence in the the half-life of the fusion protein used to the in fluorescence of the fusion protein. of the fluorescence a that the half-life of the fusion protein is 2 h. had of fluorescence to the the the half-life of the EGFP fusion protein and its fluorescence, the EGFP and EGFP-MODC-(422–461) transfected used for also used for Western blot analysis with a in the was to EGFP and the fusion protein. in the of EGFP was However, the EGFP-MODC-(422–461) fusion protein was as by a of the fusion protein was 2 of We that the half-life of the EGFP-MODC-(422–461) fusion protein is h. that the fluorescence decay of the fusion protein with its protein The fusion protein was blot analysis of protein of EGFP and used for The to and a EGFP and the EGFP fusion protein detected with a the of PEST amino acids to protein degradation in dEGFP, mutated and of dEGFP, as as the amino to degradation by the in fluorescence The for fusion mutants in We that the degradation was not to the of PEST was by such as the of the amino the protein, of the amino acids and the of the 2 that the and of the PEST sequence to protein in of the PEST sequence of EGFP-MODC-(422–461) and the of the analysis of EGFP, and in transfected was in the as with for and and for fluorescence by The fluorescent time as a of in a can be used as a transcription it to of NFκB binding sequence and the The was transfected for monitoring NFκB The time course induction with kinetics is similar to used reporter. of and 2 was and The that can be used as a reporter to transcription unmodified EGFP, when used as the reporter for did not to the This result be of a of the stable protein in the cells. unlike dEGFP, a rapid stable EGFP be used as a reporter for the of NFκB transfected by the reporter the with The for analysis with of fluorescence was EGFP, is an protein not require a to use it to the transcription induction and NFκB translocation the to the nucleus. with with for and and with NFκB and with a in induction was with and the translocation of NFκB with NFκB was in the of rapid translocation of NFκB the to the nucleus. This was was not detected this 2 NFκB had in the and the induction of NFκB the to the and the induction of was h. The that the translocation of NFκB is in response to and that of the NFκB was to the h. This translocation of NFκB in its in the of transcription induction of detection of NFκB localization and induction of transfected by the with for and h. NFκB translocation was with and induction was by NFκB translocation and induction of detected The of MODC, amino acids ornithine decarboxylase degradation in This a PEST sequence amino acids the PEST domain can also EGFP it to the C-terminal end of EGFP to This fusion was a expression The fluorescence of the fusion protein was a fluorescence it was in cells. that the fluorescence of EGFP-MODC-(422–461) is similar to EGFP with in the tested the of the of to EGFP degradation in The was transfected cells. the with for and and the in fluorescence of the transfected was by fluorescence The fluorescence of the fusion protein in the as was that the fusion protein is a with fluorescence had by The that the half-life of the fusion protein is h. did not a in the fluorescence in the the half-life of the fusion protein used to the in fluorescence of the fusion protein. of the fluorescence a that the half-life of the fusion protein is 2 h. had of fluorescence to the the the half-life of the EGFP fusion protein and its fluorescence, the EGFP and EGFP-MODC-(422–461) transfected used for also used for Western blot analysis with a in the was to EGFP and the fusion protein. in the of EGFP was However, the EGFP-MODC-(422–461) fusion protein was as by a of the fusion protein was 2 of We that the half-life of the EGFP-MODC-(422–461) fusion protein is h. that the fluorescence decay of the fusion protein with its protein The fusion protein was the of PEST amino acids to protein degradation in dEGFP, mutated and of dEGFP, as as the amino to degradation by the in fluorescence The for fusion mutants in We that the degradation was not to the of PEST was by such as the of the amino the protein, of the amino acids and the of the 2 that the and of the PEST sequence to protein in was in the as with for and and for fluorescence by The fluorescent time as a of can be used as a transcription it to of NFκB binding sequence and the The was transfected for monitoring NFκB The time course induction with kinetics is similar to used reporter. of and 2 was and The that can be used as a reporter to transcription unmodified EGFP, when used as the reporter for did not to the This result be of a of the stable protein in the cells. unlike dEGFP, a rapid stable EGFP be used as a reporter for the of dEGFP, EGFP, is an protein not require a to use it to the transcription induction and NFκB translocation the to the nucleus. with with for and and with NFκB and with a in induction was with and the translocation of NFκB with NFκB was in the of rapid translocation of NFκB the to the nucleus. This was was not detected this 2 NFκB had in the and the induction of NFκB the to the and the induction of was h. The that the translocation of NFκB is in response to and that of the NFκB was to the h. This translocation of NFκB in its in the of transcription induction of destabilized EGFP by the degradation domain of to the of The fusion protein is unstable in the presence of with indicated that the half-life of is 2 h. Western blot analysis indicated that the half-life was h. The in the half-life by of the presence of and of the EGFP is and with a of GFP, Western and the result in half-life Because to use GFP as a reporter the fluorescence the protein its fluorescence half-life is a We that the half-life of is 2 of that EGFP degradation in a PEST sequence amino acids to amino and and analysis and is for protein However, that degradation not correlate with the of PEST mutants such as This in a with in the of is correlated to the of in the sequence the PEST by the of PEST sequences of to protein Because it is easily EGFP is an reporter for studies of protein rapid of the rapid of allows its application studies destabilized GFP, such as studies in the its rapid in accumulation in to in the when that a stable this fusion in of unmodified EGFP be the destabilized of EGFP can be used as a reporter in the of the of as a transcription reporter to that of NFκB activation. The kinetics of time course induction of and dose response similar to of the kinetics of EGFP The for the is that of its EGFP is easily the of the induction of EGFP is that of the rapid of its accumulation in time course induction similar to was also to the induction of by not that is of as a transcription reporter. Because fluorescence can be detected in time the of it an in the such as that reporter with can be used for in and to of induction can be directly by monitoring the of the fluorescence with a of fluorescence of destabilized GFP by the to transcription transcription factor NFκB expression of a of to and response of NFκB rapid degradation of its and translocation of the NFκB the to the degradation of is a rapid The binding of NFκB can be detected degradation The degradation of is and it of the The of is also that it protein Because is the gene of it is that the of is by The not the binding of NFκB a for NFκB to induction of the that of and the NFκB is this that the of NFκB in the is also the of NFκB protein to h. Because was used as a reporter in this induction can be with the of We that the of NFκB in the is with its induction of the reporter gene use of the protein as a transcription reporter allows monitoring of transcription induction directly and its with biochemical We destabilized EGFP by the degradation domain of to the of The fusion protein is unstable in the presence of with indicated that the half-life of is 2 h. Western blot analysis indicated that the half-life was h. The in the half-life by of the presence of and of the EGFP is and with a of GFP, Western and the result in half-life Because to use GFP as a reporter the fluorescence the protein its fluorescence half-life is a We that the half-life of is 2 h. The of that EGFP degradation in a PEST sequence amino acids to amino and and analysis and is for protein However, that degradation not correlate with the of PEST mutants such as This in a with in the of is correlated to the of in the sequence the PEST by the of PEST sequences of to protein Because it is easily EGFP is an reporter for studies of protein The rapid of the rapid of allows its application studies destabilized GFP, such as studies in the its rapid in accumulation in to in the when that a stable this fusion in of unmodified EGFP be the destabilized of EGFP can be used as a reporter in the of We the of as a transcription reporter to that of NFκB activation. The kinetics of time course induction of and dose response similar to of the kinetics of EGFP The for the is that of its EGFP is easily the of the induction of EGFP is that of the rapid of its accumulation in time course induction similar to was also to the induction of by not that is of as a transcription reporter. Because fluorescence can be detected in time the of it an in the such as that reporter with can be used for in and to of induction can be directly by monitoring the of the fluorescence with a of fluorescence of destabilized GFP by the to transcription The transcription factor NFκB expression of a of to and response of NFκB rapid degradation of its and translocation of the NFκB the to the degradation of is a rapid The binding of NFκB can be detected degradation The degradation of is and it of the The of is also that it protein Because is the gene of it is that the of is by The not the binding of NFκB a for NFκB to induction of the that of and the NFκB is this that the of NFκB in the is also the of NFκB protein to h. Because was used as a reporter in this induction can be with the of We that the of NFκB in the is with its induction of the reporter gene use of the protein as a transcription reporter allows monitoring of transcription induction directly and its with biochemical We for for with for and for the and and for of