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Cell-based biosensors based on intein-mediated protein engineering for detection of biologically active signaling molecules Hyunjin Jeon, Euiyeon Lee, Dahee Kim, Minhyung Lee, Jeahee Ryu, Chungwon Kang, Soyoun Kim, and Youngeun Kwon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01481 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 22, 2018
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Analytical Chemistry
Cell-based biosensors based on intein-mediated protein engineering for detection of biologically active signaling molecules Hyunjin Jeon, Euiyeon Lee, Dahee Kim, Minhyung Lee, Jaehee Ryu, Chungwon Kang, Soyoun Kim, and Youngeun Kwon* Department of Biomedical Engineering (BK21 plus), Dongguk University, Seoul 04620, Korea * To whom the correspondence should be addressed:
[email protected] KEYWORDS Cell-based biosensor, split inteins, protein splicing, protein cleavage, fluorescence-translocation
ABSTRACT: Live cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Genetically encoded sensor cells often utilize bimolecular fluorescence complementation or fluorescence resonance energy transfer to build a reporter unit that suffers from non-specific signal activation at high concentrations. Here, we designed genetically encoded sensor cells that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. In this work, the target molecules or the external stimuli activated intein-mediated reactions, which resulted in activation of the fluorophoreconjugated signal peptide. This approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. We could also avoid the necessity of designing split reporters to refold into active structures upon reconstitution. These live cell-based sensors were able to detect biologically active signaling molecules, such as Ca2+ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca2+ stimuli and the glucocorticoid receptor agonist, Dexamethasone. These live cell-based sensing systems hold large potential for applications such as drug screening and toxicology studies, which require functional information about targets.
INTRODUCTION Biosensors are analytical devices designed to detect the presence of biomolecules or biologically active molecules. Various types of biosensors, including molecular biosensors as well as cell- and tissue-based sensors, are currently available1-11. Among these sensors, molecular biosensors are the most popular because they offer the most sensitive sensing platform with a quick response time1-3. However, molecular sensors can provide only analytical information, such as if and how much of the target molecules
are present in a sample. Alternatively, whole cell-based sensors are attracting a great deal of attention as a screening platform for biologically active molecules because they enable the monitoring of functions and interactions of target biomolecules in their native environment, where diverse biological molecules co-exist, including compounds with similar functions4-16. Functional information about target molecules obtained using mammalian cells is especially useful for applications such as pharmacology and cell physiology.
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Scheme 1. Schematic description of fluorescence translocation sensor cells using intein-mediated reactions. (a) The split intein-mediated conditional protein splicing (CPS) reaction is used to reconstitute the signal peptide. The signaling molecule induces the heterodimerization of sensor proteins to trigger the CPS. The split signal peptide is reconstituted via a stable amide bond and regains its activity. The fluorescent reporter is translocated by the activated signal peptide and reports the presence of the signaling molecule. (b) The split inteinmediated conditional protein cleavage (CPC) reaction is used to activate the signal peptide. The signaling molecule induces the nuclear localization of a sensor protein to trigger CPC. The C-terminus of the signal peptide is exposed via the specific cleavage reaction and the signal peptide is activated. The fluorescent reporter (Fluorescent protein, F) is translocated by the activated signal peptide and reports the presence of the signaling molecule.
Cell-based sensors are often fabricated in the form of genetically encoded biosensors using native receptors or enzymes as a molecular recognition component in living cells6-12. The events recognized by these recognition units, i.e., the presence of target molecules or cellular signaling induced by target molecules, must be converted to a readable output for reporting11. Optical sensing is the detection method of choice because it allows non-invasive and real-time monitoring of cellular events with a superb spatiotemporal resolution12. For optical sensing, autofluorescent proteins (AFPs) and bioluminescent proteins are often used as reporter systems in the form of fluorescence activation/deactivation, fluorescent/bioluminescent resonance energy transfer (FRET/BRET), and bimolecular fluorescence/luminescence complementation systems (BiFC)13-16. While the FRET- and BiFC- based detection offers a useful tool for biosensing, there are a couple of drawbacks. First, considerable design effort is required for efficient energy transfer or fluorescence activation13,14. Second, there could be high background signals when high concentration of fluorescent proteins are coexpressed in cells15,16. These reporting systems are often interfaced with intein-mediated protein engineering approaches to enhance their performance17-21. A selfprocessing protein, intein, and the intein-mediated protein engineering has become an important tool in biotechnology and offers solutions for some technical hurdles in the fabrication of genetically encoded cell-based sensors22-26. Especially, the split inteins that can conduct conditional protein splicing (CPS) have been popularly used23-26. In these applications, the fusions of biological recognition components and split inteins are stimulated by biological targets to reconstitute split-reporter pro-
teins, such as luciferases and AFPs, via protein transsplicing (PTS). Intein-mediated protein reconstitution offers an improved alternative to previous FRET and BiFC-based approaches because it enables covalent reconstitution of reporter proteins, resulting in enhanced signal intensity, increased target sensitivity, and improved biostability. While useful, one major drawback is that inteinmediated reconstitution of reporter proteins still requires an optimal combination structure that can spontaneously fold into a functional structure upon reconstitution27,28. In addition, split AFP or luciferases can generate falsepositive signals in the absence of stimuli because the split fragments of reporter proteins have considerable binding affinity to each other11. One of the major characteristics of intein-mediated reactions is the ability to form and break specific amide bonds29-31. In this work, we designed a live cell-based sensing system by fully appreciating these merits. The key to our approach is the activation of signal peptides and consequent fluorescence-translocation mediated by CPS or conditional protein cleavage (CPC) reactions with the target molecule as an activation switch. CPS was used to conjugate a pair of split-signal peptides, while CPC was used to expose the C-terminus of the signal peptide for activation. Unlike previous systems, split signal peptides have low binding affinity to each other and cannot be activated without covalent conjugation. Moreover, the reconstituted signal peptide does not require refolding into a specific tertiary structure for activation. We expect that this approach will provide a novel method to design a reporter unit in cell-based sensors because it eliminates false-positive signals derived from non-specific recombi-
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Analytical Chemistry
nation and provides a way to circumvent troublesome refolding of reconstituted proteins.
EXPERIMENTAL METHODS Figure 1. Domain architecture of the fusion proteins used in this study.
General. General chemicals of the best grade available were supplied by Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific (Pittsburgh, PA, USA). DNA primers were ordered from MBiotech (Hanam, Korea). Restriction enzymes were purchased from Elpis Biotech (Daejeon, Korea) and New England Biolabs (MA, USA). General cell culture reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA) and Welgene (Daegu, Korea). SDS-PAGE and Western blotting analyses were performed following standard protocols. Confocal fluorescence images were obtained using an Eclipse Ti (Nikon Instruments, Tokyo, Japan) with excitation wavelengths of 358 nm, 488 nm, 594 nm and corresponding emission filters. The fluorescence intensity was measured using Nikon NIS-Element BR 4.60 software. Plasmid construction. DNA cloning was carried out according to standard protocols. All constructed plasmids were confirmed by DNA sequencing, amplified using the E. coli strain DH5α and used for protein expression. The cDNA encoding the N-terminal domain of Saccharomyces cerevisiae VMA intein (VMAN) in pET28a was modified by introducing the cDNAs encoding the His6 tag and the Nterminal fragment of the nuclear localization signal (NLSN, KRPAATKKA) to the N-terminus between NcoI and NheI sites and then the cDNA encoding calmoduline (CaM) was inserted to the C-terminus between SalI and XhoI to create the construct 1. The cDNA encoding the Cterminal domain of the VMA intein (VMAC) in pET28a was modified by introducing cDNA encoding the Cterminal fragment of NLS (NLSC, GQAKKKKLD) and mCherry to the C-terminus between NheI and XhoI sites. The resulting construct was modified by inserting the cDNA encoding calmodulin binding domain (CaMBD) on the N-terminus between NcoI and NdeI sites to create the construct 2. The cDNA encoding mCherry in pBI-CMV1 was modified by introducing the cDNA encoding the CAAX tag (KHKEKMSKDGKKKKKKSKTKCVIM) originating from KRas-4B32, Nostoc punctiforme DnaE N-intein
and the NLS peptide (NpuN), (KRPAATKKAGQAKKKKLD)33 originating from Xenopus laevis nucleoplasmin to the C-terminus between ApaI and XbaI sites to create construct 3. The glucocorticoid receptor (GR) sequence from the human pituitary gland QUICK-Clone cDNA library was inserted into the Cterminus of Npu DnaE C-intein (NpuC) in pBI-CMV1 between MluI and EcoRV sites to create construct 4. The NpuN sequence was introduced between EcoRI and HindIII sites of pMAL-c2 to create construct 5. The GST sequence is cloned between HindIII and XhoI in pET28a vector and modified by inserting the sequence encoding NpuC and Flag-Flag tag on the N-terminus between NocI and HindIII sites to create construct 6. Construct 7 was prepared by the same approach but with modified NpuC (mNpuC) instead of NpuC. The cDNA encoding mCherry in pET28a was modified by inserting the sequence encoding CAAX tag and a stop codon to the C-terminus between NdeI and EcoRI sites to create construct 8. Construct 9 was prepared by the same approach as for construct 8 but with a stop codon. Construct 10 was prepared using the same approach as for construct 8 but with cDNA encoding GR instead of NpuC. PTS/protein trans-cleavage (PTC) using purified proteins. The target proteins were expressed using BL21(DE3). The bacterial cells were lysed in PTS buffer (TS buffer, 50 mM Tris/HCl, 300 mM NaCl, 1 mM EDTA, pH 7.0). The crude supernatants containing fusion proteins were mixed to have equal amounts of the N- and Cintein fusion proteins, and the reaction mixture was allowed to react at 37°C in the presence of 50 mM DTT. The reaction mixture was analyzed by SDS-PAGE. Splicing/cleavage product formation was monitored by Western blot analysis using an anti-MBP antibody. Live cell imaging for Ca2+ sensing. For live cell imaging, HeLa cells were grown in 35 mm imaging dishes. The cells were transiently transfected using pBI-CMV1 vector encoding fusion proteins 1 and 2. Gene expression was allowed to proceed at 37°C for 40 h; then, cell-based sensing assays were started. For Ca2+ stimulation, cells were treated with 20 nM ionomycin or 10 µM Histamine for 3 h in media containing 5 mM CaCl2. After Ca2+ stimulation, cell nuclei were stained using Syto9 (2.5 µM) for 10 min, washed twice using PBS, and imaged using confocal fluorescence microscopy. In average, 30 cells were analyzed in each experiment. Live cell imaging for hormone sensing. For live cell imaging, HeLa cells were grown in 35 mm imaging dishes. The cells were transiently transfected using pBI-CMV1 vector encoding fusion proteins 3 and 4. Gene expression was allowed to proceed at 37°C for 40 h, at which point cell-based sensing assays were started. For hormonal stimulation, cells were individually treated with 1 µM cortisol or analogue in media containing 1% FBS. Twelve hours after stimulation, cell nuclei were stained using Hoechst 33342 dye and imaged using confocal fluorescence microscopy. In average, 30 cells were analyzed in each experiment.
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Figure 2. Detection of direct Ca2+ stimulation using sensor cells based on conditional protein splicing reaction. (a) Sensor cells coexpressing 1 and 2 show fluorescence from mCherry in the cytosol prior to stimulation (row 1). The fluorescence signal travels to the nucleus when the sensor cells are challenged with CaCl2 and ionomycin (row 2). The control cells expressing only protein 2 do not respond to Ca2+ stimulation (row 3). The nucleus was stained with Syto9 (green). Scale bar = 20 µm. (b) The fluorescence signals throughout the whole cell are quantitatively analyzed. (c) The ratios of red fluorescence intensity from the nucleus to the cytoplasm are calculated. Data were analyzed by one-way ANOVA using Turkey’s multiple comparison test. ***p
