BACKGROUND
real-time sequential monitoring of recombinant protein production is advantageous over single-event, terminal monitoring that requires destruction of expressing cells in vitro or the ex vivo analysis of clinical samples.
for example, transfected cell cultures may require lysis for detection of recombinant proteins of interest through polyacrylamide gel electrophoresis, western blots, elisa, indirect fluorescent antibody assay or other methods.
these detection methods are time-consuming, costly and often require protein-specific antibody reagents.
monitoring in vivo expression of recombinant proteins is more problematic.
it requires invasive sampling at fewer time points, or terminal procedures, as well as protein-specific reagents.
linking expression of a recombinant protein of interest to an easily detectable, secreted biomarker in a single open reading frame would allow for rapid, quantitative, and sequential monitoring of all proteins within the transcriptional unit.
moreover, using a secreted biomarker would be a useful tool for quantitating in vivo recombinant protein expression independent from host immune responses.
the gaussia princeps luciferase  is a naturally secreted luciferase that catalyzes oxidation of the substrate coelenterazine to produce an intense luminescent burst  <cit> .
gluc is readily quantifiable in clinical samples  within a linear detection range .
the luminescent output of wild-type gluc is enhanced by mutation of two amino acid residues, f89w and i90l, resulting in a super-luminescent gluc variant  with a peak emission wavelength of 481 nm  <cit> .
we sought to use secreted gluc  as a general biomarker to monitor overall expression of recombinant proteins from a single transcriptional unit.
gluc is non-native to the mammalian system.
this allows for more definitive quantification than other enzymatic biomarkers, such as secreted embryonic alkaline phosphatase, which can have innate levels in vivo  <cit> .
production of multiple recombinant proteins from a single open-reading frame has been previously accomplished through use of proteolytic cleavage, self-processing peptides, multiple internal ribosome entry sites , and other mechanisms  <cit> .
foot-and-mouth disease virus  encodes a nonstructural 2a translational interrupter which induces ribosome skipping causing the separation of the fmdv p <dig> and p <dig> polyproteins in a non-proteolytic manner  <cit> .
the efficiency of fmdv 2a-mediated translational interruption is amino acid sequence dependent, and its activity is enhanced when the additional sequence derived from the c-terminus of the fmdv 1d  protein is included  <cit> .
fmdv 2a-mediated polyprotein separation is nearly 100% efficient and produces a constant 1: <dig> yield of proteins on either side of the fmdv 2a sequence  <cit> .
therefore, a fusion of gluc and fmdv 2a sequences potentially provides a mechanism to directly correlate yields of transcriptionally-linked recombinant proteins by assaying for secreted gluc activity.
such an assay would enable sequential, non-destructive sampling and normalization among test samples.
we report the production and evaluation of six distinct chimeras of gluc or sgluc  variants with the fmdv 2a translational interrupter on either the n- or c-terminus within a single open reading frame, including two novel gluc/sgluc variants with a deleted methionine start codon.
we also evaluated the ability of one chimera to function as the 3′ terminus of a transgene encoding a fmdv p1-2a-3c cassette known to produce vlps.
RESULTS
design of six bicistronic gluc/sgluc constructs
a total of six bicistronic gluc/sgluc constructs were evaluated for retention of secretion and ability to luminesce .
to facilitate efficient translational interruption in bicistronic constructs, we used a modified fmdv 2a sequence identified as Δ1d2a  <cit>  consisting of the  <dig> c-terminal amino acids of vp <dig> , and the defined  <dig> amino acid 2a sequence with a c-terminal proline   <cit> .
four bicistronic constructs had the Δ1d2a sequence inserted on either the n- or c-terminus of either gluc/sgluc.
we found that the methionine normally at position  <dig> was dispensable for translation initiation when the Δ1d2a was positioned on the n-terminus of gluc/sgluc .
consequently, the final two bicistronic constructs were composed of gluc/sgluc templates with an n-terminal Δ1d2a sequence and a deletion of the position  <dig> methionine within the luciferase  .fig.
<dig>
a construct layouts of bicistronic templates evaluated.
b nucleotide and amino acid sequences of Δ1d2a translational interrupter
fig.
<dig>
a western blot probed with anti-gluc antibody showing roughly equalized loading.
b cell culture media luciferase readings for bicistronic templates using volumes determined by western blotting.
rfus, relative fluorescent units
luciferase activity in cell culture
addition of the c-terminal Δ1d2a sequence in gluc-Δ1d2a and sgluc-Δ1d2a constructs resulted in a molecular weight increase of resulting proteins as visualized in western blots used to establish relative sample loading prior to quantifying luciferase activities .
luciferase activity was present in all samples except the histone negative control confirming that addition of the Δ1d2a sequence on either the n- or c-terminus of the luciferase did not prevent secretion or luminescence .
relative sample loading was used to account for the possibility that the addition of the Δ1d2a to gluc/sgluc may alter luminescent intensities.
by establishing relative sample loading using an anti-gluc polyclonal antibody we were able to observe that the gluc-Δ1d2a construct displayed  <dig> and 48% reductions in luminescent output versus the unmodified gluc and Δ1d2a-gluc constructs .
the sgluc-Δ1d2a construct exhibited a  <dig> and 68% reduction relative to Δ1d2a-sgluc and sgluc constructs .
deletion of the gluc/sgluc start codon, Δ1m, from n-terminal chimeras did not prevent sgluc secretion and resulted in an 18% reduction compared to Δ1d2a-gluc Δ1m and a 6% increase when compared to Δ1d2a-sgluc.
luciferase activity in cell lysis buffer
despite previous reports identifying the sgluc variant as emitting approximately 10-fold higher luminescence than gluc from lysed cells  <cit> , we measured a 36% average increase in luminescence in cell culture media .
to determine if the choice of cell lysis buffer affected the luminescence output between gluc and sgluc, we combined harvested cell media from each variant with one of two common cell lysis buffers , and measured luciferase levels .
the sgluc and gluc constructs lost > <dig> and >80% activity when diluted in either cell lysis buffer as compared to control cell culture media, but the sgluc construct retained approximately four-fold more luciferase activity than the gluc construct in either cell lysis buffer.fig.
<dig> percentage of total sample luciferase activity retained when mixed with one of two cell lysis buffers, lclb  or m-per, as compared to cell culture media designated as 100%
a biomarker for expression in fmdv vlp producing constructs
to determine if the transcriptionally-linked luciferase affected the ability of a fmdv transgene to produce virus-like particles , we added the Δ1d2a-sgluc chimera to the c-terminus of a plasmid vector, p1-2a-3c, capable of producing fmdv vlps in transfected cell cultures .
luciferase activity was readily detected in media from lfbk-αvβ <dig> cells transfected with the fmdv p1-2a-3c-Δ1d2a-sgluc construct .fig.
<dig> construct layouts along with subsequently expressed and processed fmdv proteins used for the evaluation of vlp formation while utilizing the Δ1d2a-sgluc sequence
fig.
<dig> luciferase activity from transfected lfbk-αvβ <dig> cell culture media.
rfus, relative fluorescent units
using immunoelectron microscopy  we observed electron-lucent arrays of 25–30 nm diameter fmdv vlps in the cytoplasm of lfbk-αvβ <dig> cells transfected with either p1-2a-3c or p1-2a-3c-Δ1d2a-sgluc plasmids .
immunogold labeled fmdv conformation dependent monoclonal antibody   localized to these array structures .
immunofluorescent assays with two additional non-conformation dependent fmdv-specific mabs, 6hc <dig> and 12fe <dig>  further validated serotype-specific expression of fmdv proteins in transfected cells .fig.
<dig> immunoelectron microscopy images using immunogold coated with monoclonal antibody f <dig> to localize fmdv vp <dig> in the virus-like particle arrays of lfbk-αvβ <dig> cells transfected with construct p1-2a-3c-Δ1d2a-sgluc at a 5000×  and b  <dig> ×  as well as construct p1-2a-3c at c 5000×  and d  <dig> ×
fig.
<dig> immunofluorescent assay with fmdv specific monoclonal antibodies 6hc <dig>  12fe <dig> and f <dig> on lfbk-αvβ <dig> cells transfected with constructs encoding either sgluc, p1-2a-3c, or p1-2a-3c-Δ1d2a-sgluc
discussion
we produced gluc/sgluc Δ1d2a luciferase chimeras that retained both secretory and luciferase activity, and demonstrated use of these chimeras as tools to quantitate transgene expression through sequential, non-destructive sampling of all culture media.
in our system, the sgluc variant showed enhanced luminescence over gluc, but at lower levels than previously reported  <cit> .
we showed that this difference is influenced by the choice of cell lysis buffer, and may explain the differences in luciferase intensities observed between our results and those previously reported utilizing secretion-deficient mutants harvested from lysed cells  <cit> .
the reduction of luciferase activity in cell lysis buffer may be due to the presence of denaturing conditions which may have more impact on gluc than on sgluc.
constructs containing a c-terminal Δ1d2a showed less luminescence despite having similar levels of protein tested, as determined by western blot.
these data suggest that the luminescent activity of these chimeras was inhibited compared to n-terminal and gluc/sgluc constructs.
we produced a plasmid encoding the fmdv p <dig>  2a, 3c genes in-frame with the Δ1d2a-sgluc sequence on the 3′ terminus.
this marker-vaccine construct retained the ability to produce assembled fmdv vlps along with a concomitant readout of luciferase activity in culture media as an easily quantifiable marker of fmdv protein production.
notably, the vlp crystalline arrays produced in this system appeared to be identical to those previously reported from cells infected with wildtype fmdv  <cit> .
the conformation dependent f <dig> mab will only interact with vp <dig> in the proper confirmation, such as when it is contained in a vlp.
as fmdv vlps form and become localized within arrays it results in the localization of f <dig> binding to these arrays as observed in iem.
rapid quantitation of recombinant protein levels is a critical tool for molecular biology.
the system described here provides a direct correlate to quantitate recombinant proteins produced in various matrices, including clinical samples, without the need for antigen-specific reagents.
for development of recombinant vaccines, this system may provide a mechanism to monitor recombinant antigen production in vivo following vaccination, determine vaccine vector expression kinetics, optimize delivery methods, and correlate the amount of recombinant antigen produced with host immune response, and protection from infection.
lastly, this reporter system may be widely applicable to monitor transgene expression for a variety of biomedical purposes including gene therapy, drug screening, vaccine production, and bio-manufacturing.
CONCLUSIONS
we describe the use of gaussia luciferase chimeras as a reporter system for quantifying recombinant gene expression.
gluc/sgluc variants with the fmdv Δ1d2a sequence placed on either the n- or c- terminus retained secretory and luciferase activity that enabled rapid, sequential quantitation of transgene expression through non-destructive sampling without need for protein-specific detection reagents.
placing the Δ1d2a-sgluc sequence downstream of an fmdv vlp production cassette resulted in both vlp production and luciferase activity, thereby demonstrating the practical application of this technology to recombinant vaccine development.

 real-time sequential monitoring of recombinant protein production is advantageous over single-event, terminal monitoring that requires destruction of expressing cells in vitro or the ex vivo analysis of clinical samples. for example, transfected cell cultures may require lysis for detection of recombinant proteins of interest through polyacrylamide gel electrophoresis, western blots, elisa, indirect fluorescent antibody assay or other methods. these detection methods are time-consuming, costly and often require protein-specific antibody reagents. monitoring in vivo expression of recombinant proteins is more problematic. it requires invasive sampling at fewer time points, or terminal procedures, as well as protein-specific reagents. linking expression of a recombinant protein of interest to an easily detectable, secreted biomarker in a single open reading frame would allow for rapid, quantitative, and sequential monitoring of all proteins within the transcriptional unit. moreover, using a secreted biomarker would be a useful tool for quantitating in vivo recombinant protein expression independent from host immune responses. the gaussia princeps luciferase is a naturally secreted luciferase that catalyzes oxidation of the substrate coelenterazine to produce an intense luminescent burst. gluc is readily quantifiable in clinical samples within a linear detection range. the luminescent output of wild-type gluc is enhanced by mutation of two amino acid residues, f89w and i90l, resulting in a super-luminescent gluc variant with a peak emission wavelength of 481 nm. we sought to use secreted gluc as a general biomarker to monitor overall expression of recombinant proteins from a single transcriptional unit. gluc is non-native to the mammalian system. this allows for more definitive quantification than other enzymatic biomarkers, such as secreted embryonic alkaline phosphatase, which can have innate levels in vivo. production of multiple recombinant proteins from a single open-reading frame has been previously accomplished through use of proteolytic cleavage, self-processing peptides, multiple internal ribosome entry sites, and other mechanisms. foot-and-mouth disease virus encodes a nonstructural 2a translational interrupter which induces ribosome skipping causing the separation of the fmdv p and p polyproteins in a non-proteolytic manner. the efficiency of fmdv 2a-mediated translational interruption is amino acid sequence dependent, and its activity is enhanced when the additional sequence derived from the c-terminus of the fmdv 1d protein is included. fmdv 2a-mediated polyprotein separation is nearly 100% efficient and produces a constant 1: yield of proteins on either side of the fmdv 2a sequence. therefore, a fusion of gluc and fmdv 2a sequences potentially provides a mechanism to directly correlate yields of transcriptionally-linked recombinant proteins by assaying for secreted gluc activity. such an assay would enable sequential, non-destructive sampling and normalization among test samples. we report the production and evaluation of six distinct chimeras of gluc or sgluc variants with the fmdv 2a translational interrupter on either the n- or c-terminus within a single open reading frame, including two novel gluc/sgluc variants with a deleted methionine start codon. we also evaluated the ability of one chimera to function as the 3′ terminus of a transgene encoding a fmdv p1-2a-3c cassette known to produce vlps.  design of six bicistronic gluc/sgluc constructs a total of six bicistronic gluc/sgluc constructs were evaluated for retention of secretion and ability to luminesce. to facilitate efficient translational interruption in bicistronic constructs, we used a modified fmdv 2a sequence identified as Δ1d2a consisting of the c-terminal amino acids of vp, and the defined amino acid 2a sequence with a c-terminal proline. four bicistronic constructs had the Δ1d2a sequence inserted on either the n- or c-terminus of either gluc/sgluc. we found that the methionine normally at position was dispensable for translation initiation when the Δ1d2a was positioned on the n-terminus of gluc/sgluc. consequently, the final two bicistronic constructs were composed of gluc/sgluc templates with an n-terminal Δ1d2a sequence and a deletion of the position methionine within the luciferase.fig. a construct layouts of bicistronic templates evaluated. b nucleotide and amino acid sequences of Δ1d2a translational interrupter fig. a western blot probed with anti-gluc antibody showing roughly equalized loading. b cell culture media luciferase readings for bicistronic templates using volumes determined by western blotting. rfus, relative fluorescent units luciferase activity in cell culture addition of the c-terminal Δ1d2a sequence in gluc-Δ1d2a and sgluc-Δ1d2a constructs resulted in a molecular weight increase of resulting proteins as visualized in western blots used to establish relative sample loading prior to quantifying luciferase activities. luciferase activity was present in all samples except the histone negative control confirming that addition of the Δ1d2a sequence on either the n- or c-terminus of the luciferase did not prevent secretion or luminescence. relative sample loading was used to account for the possibility that the addition of the Δ1d2a to gluc/sgluc may alter luminescent intensities. by establishing relative sample loading using an anti-gluc polyclonal antibody we were able to observe that the gluc-Δ1d2a construct displayed and 48% reductions in luminescent output versus the unmodified gluc and Δ1d2a-gluc constructs. the sgluc-Δ1d2a construct exhibited a and 68% reduction relative to Δ1d2a-sgluc and sgluc constructs. deletion of the gluc/sgluc start codon, Δ1m, from n-terminal chimeras did not prevent sgluc secretion and resulted in an 18% reduction compared to Δ1d2a-gluc Δ1m and a 6% increase when compared to Δ1d2a-sgluc. luciferase activity in cell lysis buffer despite previous reports identifying the sgluc variant as emitting approximately 10-fold higher luminescence than gluc from lysed cells, we measured a 36% average increase in luminescence in cell culture media. to determine if the choice of cell lysis buffer affected the luminescence output between gluc and sgluc, we combined harvested cell media from each variant with one of two common cell lysis buffers, and measured luciferase levels. the sgluc and gluc constructs lost > and >80% activity when diluted in either cell lysis buffer as compared to control cell culture media, but the sgluc construct retained approximately four-fold more luciferase activity than the gluc construct in either cell lysis buffer.fig. percentage of total sample luciferase activity retained when mixed with one of two cell lysis buffers, lclb or m-per, as compared to cell culture media designated as 100% a biomarker for expression in fmdv vlp producing constructs to determine if the transcriptionally-linked luciferase affected the ability of a fmdv transgene to produce virus-like particles, we added the Δ1d2a-sgluc chimera to the c-terminus of a plasmid vector, p1-2a-3c, capable of producing fmdv vlps in transfected cell cultures. luciferase activity was readily detected in media from lfbk-αvβ cells transfected with the fmdv p1-2a-3c-Δ1d2a-sgluc construct.fig. construct layouts along with subsequently expressed and processed fmdv proteins used for the evaluation of vlp formation while utilizing the Δ1d2a-sgluc sequence fig. luciferase activity from transfected lfbk-αvβ cell culture media. rfus, relative fluorescent units using immunoelectron microscopy we observed electron-lucent arrays of 25–30 nm diameter fmdv vlps in the cytoplasm of lfbk-αvβ cells transfected with either p1-2a-3c or p1-2a-3c-Δ1d2a-sgluc plasmids. immunogold labeled fmdv conformation dependent monoclonal antibody localized to these array structures. immunofluorescent assays with two additional non-conformation dependent fmdv-specific mabs, 6hc and 12fe further validated serotype-specific expression of fmdv proteins in transfected cells.fig. immunoelectron microscopy images using immunogold coated with monoclonal antibody f to localize fmdv vp in the virus-like particle arrays of lfbk-αvβ cells transfected with construct p1-2a-3c-Δ1d2a-sgluc at a 5000× and b × as well as construct p1-2a-3c at c 5000× and d × fig. immunofluorescent assay with fmdv specific monoclonal antibodies 6hc 12fe and f on lfbk-αvβ cells transfected with constructs encoding either sgluc, p1-2a-3c, or p1-2a-3c-Δ1d2a-sgluc discussion we produced gluc/sgluc Δ1d2a luciferase chimeras that retained both secretory and luciferase activity, and demonstrated use of these chimeras as tools to quantitate transgene expression through sequential, non-destructive sampling of all culture media. in our system, the sgluc variant showed enhanced luminescence over gluc, but at lower levels than previously reported. we showed that this difference is influenced by the choice of cell lysis buffer, and may explain the differences in luciferase intensities observed between our results and those previously reported utilizing secretion-deficient mutants harvested from lysed cells. the reduction of luciferase activity in cell lysis buffer may be due to the presence of denaturing conditions which may have more impact on gluc than on sgluc. constructs containing a c-terminal Δ1d2a showed less luminescence despite having similar levels of protein tested, as determined by western blot. these data suggest that the luminescent activity of these chimeras was inhibited compared to n-terminal and gluc/sgluc constructs. we produced a plasmid encoding the fmdv p 2a, 3c genes in-frame with the Δ1d2a-sgluc sequence on the 3′ terminus. this marker-vaccine construct retained the ability to produce assembled fmdv vlps along with a concomitant readout of luciferase activity in culture media as an easily quantifiable marker of fmdv protein production. notably, the vlp crystalline arrays produced in this system appeared to be identical to those previously reported from cells infected with wildtype fmdv. the conformation dependent f mab will only interact with vp in the proper confirmation, such as when it is contained in a vlp. as fmdv vlps form and become localized within arrays it results in the localization of f binding to these arrays as observed in iem. rapid quantitation of recombinant protein levels is a critical tool for molecular biology. the system described here provides a direct correlate to quantitate recombinant proteins produced in various matrices, including clinical samples, without the need for antigen-specific reagents. for development of recombinant vaccines, this system may provide a mechanism to monitor recombinant antigen production in vivo following vaccination, determine vaccine vector expression kinetics, optimize delivery methods, and correlate the amount of recombinant antigen produced with host immune response, and protection from infection. lastly, this reporter system may be widely applicable to monitor transgene expression for a variety of biomedical purposes including gene therapy, drug screening, vaccine production, and bio-manufacturing.  we describe the use of gaussia luciferase chimeras as a reporter system for quantifying recombinant gene expression. gluc/sgluc variants with the fmdv Δ1d2a sequence placed on either the n- or c- terminus retained secretory and luciferase activity that enabled rapid, sequential quantitation of transgene expression through non-destructive sampling without need for protein-specific detection reagents. placing the Δ1d2a-sgluc sequence downstream of an fmdv vlp production cassette resulted in both vlp production and luciferase activity, thereby demonstrating the practical application of this technology to recombinant vaccine development. 
we sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2a translational interrupter sequence. we demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs. the gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. we also measured the ability of another polycistronic plasmid vector with a 2a-luciferase sequence placed downstream of the foot-and-mouth disease virus p and 3c protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. incorporation of the 2a-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. gaussia luciferasefoot-and-mouth disease virus2abicistronicpolycistronicbiomarkervirus-like particleshttp://dx.doi.org/ /100000180u.s. we produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2a sequence placed either n-terminal or c-terminal to the gaussia princeps luciferase gene. two plasmids included novel gaussia princeps luciferase variants with the position methionine deleted. placing a foot-and-mouth disease virus 2a translational interrupter sequence on either the n- or c-terminus of the gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins.
 the gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. we sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2a translational interrupter sequence.  we produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2a sequence placed either n-terminal or c-terminal to the gaussia princeps luciferase gene. two plasmids included novel gaussia princeps luciferase variants with the position methionine deleted. placing a foot-and-mouth disease virus 2a translational interrupter sequence on either the n- or c-terminus of the gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. we also measured the ability of another polycistronic plasmid vector with a 2a-luciferase sequence placed downstream of the foot-and-mouth disease virus p and 3c protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. incorporation of the 2a-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles.  we demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs. keywords gaussia luciferasefoot-and-mouth disease virus2abicistronicpolycistronicbiomarkervirus-like particleshttp://dx.doi.org/ /100000180u.s. department of homeland securityde-ac05-06or23100puckette michael issue-copyright-statement© the author 2017 