BIOL 475 Biological Imaging GFP and eYFP Paper Analysis

BIOL 475 Biological Imaging GFP and eYFP Paper Analysis BIOL 475 Biological Imaging GFP and eYFP Paper Analysis NOOO PLAGIARSM ATTACHED PAPER ANALYSIS WITH QUESTIONS TO BE ANSWERED!!!! answer in full sentences and being turned into TURN IT IN cheung_hearn2003_fret.pdf paper_analysis_3_ # Eur. J. Biochem. 270, 4973–4981 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03899.x Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein–protein interaction Timothy C. Cheung and John P. Hearn Developmental Biology Research Group, Research School of Biological Sciences, The Australian National University, Canberra, Australia A new baculovirus-based ?uorescence resonance energy transfer (Bv-FRET) assay for measuring multimerization of cell surface molecules in living cells is described. It has been demonstrated that gonadotropin-releasing hormone receptor (GnRH-R) was capable of forming oligomeric complexes in the plasma membrane under normal physiological conditions. The mouse gonadotropin-releasing hormone receptor GnRH-R was used to evaluate the e?ciency and potential applications of this assay. Two chimeric constructs of GnRH-R were made, one with green ?uorescent protein as a donor ?uorophore and the other with enhanced yellow ?uorescent protein as an acceptor ?uorophore. These chimeric constructs were coexpressed in an insect cell line (BTI Tn5 B1-4) using recombinant baculoviruses. Energy transfer occurred from the excited donor to the acceptor when they were in close proximity. The association of GnRH-R was demonstrated through FRET and the ?uorescence observed using a Leica TSC-SPII confocal microscope. FRET was enhanced by the addition of a GnRH agonist but not by an antagonist. The Bv-FRET assay constitutes a highly e?cient, reliable and convenient method for measuring protein–protein interaction as the baculovirus expression system is superior to other transfection-based methods. Additionally, the same insect cell line can be used routinely for expressing any recombinant proteins of interest, allowing various combinations of molecules to be tested in a rapid fashion for protein–protein interactions. The assay is a valuable tool not only for the screening of new molecules that interact with known bait molecules, but also for con?rming interactions between other known molecules. The dimerization of cell surface molecules represents one of the most important phenomena in signal transduction because it opens a new level of understanding of the basic function and interactions of these molecules. BIOL 475 Biological Imaging GFP and eYFP Paper Analysis Many molecules that were thought to function as monomers are in fact capable of forming dimeric or oligomeric complexes, and many membrane proteins such as receptor tyrosine kinases [1,2], membrane lymphotoxin-ab ligands [3–6], receptors for growth hormone [7–10], and many G protein-coupled receptors associate as functional oligomeric complexes [11–14]. Consequently, there is an increasing demand for a reliable and convenient assay for measuring protein–protein interactions in living cells. In the past few years, a number of different ?uorescence resonance energy transfer (FRET)-based assays have been developed [15–20]. FRET is a useful method for investigating the associations of molecules. It is based on the transfer of energy from one ?uorophore (the donor) to another ?uorophore (the acceptor) that usually emits ?uorescence of a different colour. As FRET ef?ciency depends on the distance between the donor and acceptor (usually less then 100 A? apart) [21–25], it provides a useful assessment for protein–protein interaction, especially the dimerization of cell surface molecules. So far, most FRET assays performed in vivo have been performed primarily in transfected cells. A major disadvantage of the transfection-based FRET assays lies in the dif?culty of controlling the level of individual recombinant protein expression in transfected cell cultures. The expression of recombinant proteins in transient-transfected cells is in?uenced by many factors, including transfection ef?ciency of a given cell type, the quality of the DNA, the quantity of DNA taken up by the cells, the cytotoxicity of the transfection reagents, and the condition of the cells. For example, low transfection ef?ciency results in having insuf?cient cells that coexpress both donor and acceptor ?uorophores. In addition, a low level of protein expression may result in insuf?cient amounts of donor and acceptor ?uorophores located in close vicinity, reducing the probability of their interaction. Furthermore, FRET ef?ciency also depends on the ratio of coexpression between donor and acceptor ?uorophores (Table 1). To exclude artefacts Correspondence to T. C. Cheung, Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA. Fax: + 1 858 558 3525, Tel.: + 1 858 558 3500, E-mail: [email protected] Abbreviations: FRET, ?uorescence resonance energy transfer; Bv-FRET, baculovirus-based FRET; GnRH, gonadotropin-releasing hormone; GnRH-R, GnRH receptor; GFP, green ?uorescent protein; EYFP, enhanced yellow ?uorescent protein; Tn5 cells, BTI Tn5 B1-4 cells; MOI, multiplicity of infection; LTa, lymphotoxin a; LTb, lymphotoxin b; IL-2Ra, interleukin-2 receptor a-subunit. (Received 11 August 2003, revised 21 October 2003, accepted 24 October 2003) Keywords: FRET; baculovirus; membrane protein–protein interaction; dimerization; GnRH receptor. 4974 T. C. Cheung and J. P. Hearn (Eur. J. Biochem. 270) FEBS 2003 Table 1. The probability of formation of various complexes with reference to the ratios between molecules A and B. The Hardy–Weinberg law was used as the mathematical model for calculating the frequency of heterodimeric and homodimeric complexes formation. through the Cy5-labeled antibody against IL-2Ra as an acceptor ?uorophore. BIOL 475 Biological Imaging GFP and eYFP Paper Analysis ORDER NOW FOR CUSTOMIZED AND ORIGINAL NURSING PAPERS Although their assay was not designed to study protein dimerization, it demonstrated the potential application of baculovirus in the FRET-based assays [15]. GnRH-R is a member of the G protein-coupled receptors superfamily, which represents the largest grouping of cell surface receptors, mediating a wide variety of extracellular stimuli, such as light, Ca2+, odors, pheromones, peptides and proteins [35]. All G protein-coupled receptors have a common central core, which is composed of seven transmembrane domains connected be three extracellular loops and three intracellular loops [36]. Recent studies showed that GnRH-R was capable of forming multimeric complexes in the cell surface under normal physiological conditions [37,38]. It has also been shown that functionally active GnRH-R can be expressed in insect cells using recombinant baculovirus [29,30]. In the present study, the mouse GnRH-R was used to evaluate the ef?ciency of the new Bv-FRET assay. Probability (%) A : B ratios AB AA BB 1 2 3 4 5 50.0 44.4 37.5 32.0 27.8 25.0 44.4 56.2 64.0 69.4 25.0 11.2 6.3 4.0 2.8 : : : : : 1 1 1 1 1 due to aberrant donor to acceptor expression ratios and to increase the FRET signal-to-noise ratio, an optimal level of expression for both donor and acceptor is a basic requirement for FRET. Although some of the drawbacks associated with expressing recombinant proteins through transient-transfected cells could be minimized by using stable-transformed cells with the capability of coexpressing multiple recombinant proteins at a desirable level and ratio, these procedures are time consuming as well as labor intensive. Therefore, FRET assays using stable-transformed cells are unlikely to be a popular choice for many researchers. As the accuracy and sensitivity of FRET assays rely on the ability to optimize protein expression in cell culture, it is necessary to perform them using a reliable protein expression system. The baculovirus system has proven to be one of the most powerful and reliable eukaryotic protein expression systems that can be used to express functionally active recombinant proteins [26–32]. Many of the post-translational modi?cation pathways, such as phosphorylation, glycosylation, myristoylation and palmitoylation present in mammalian systems are also utilized in insect cell lines, allowing the production of recombinant protein that is functionally similar to the native mammalian protein [33,34]. Most importantly, the baculovirus system allows one to achieve a ?ne control on the level of recombinant protein expression, manipulating it by adjusting the multiplicity of infection (MOI). By combining the merits of both FRET and the baculovirus system, a new baculovirus-based FRET (BvFRET) assay was developed for detecting protein–protein interaction. This system offers all the advantages of FRET assays but overcomes the shortcomings of the transfectionbased methods. BIOL 475 Biological Imaging GFP and eYFP Paper Analysis The Bv-FRET assay has two major advantages. Firstly, it allows protein–protein interactions to be observed in living cells with confocal microscopy. Secondly, it allows direct control of the level of individual recombinant protein expression and coexpression of both donor and acceptor ?uorophores in a desirable ratio. Lundin et al. reported a FRET-based assay for measuring protein expression on the cell surface using a baculovirus expression system. Their study used europium as a donor attaching to the biotinylated cell surface of the Sf9 cells. The human interleukin-2 receptor a-subunit (IL2Ra) was also expressed on the cell surface by infecting the cells with recombinant baculoviruses. FRET was used as an assessment for protein expression on the cell surface Materials and methods Construction of expression plasmids A mouse GnRH-R/green ?uorescent protein (GFP) baculovirus expression plasmid was constructed by inserting GnRH-R cDNA in multiple cloning sites upstream of the GFP of a PVL1393 BioGreen vector (Pharmingen, San Diego, CA, USA). The mouse GnRH-R insert was synthesized by PCR using Pfu DNA polymerase (Promega, Madison, WI, USA) and mouse GnRH-R cDNA (generous gift of M. Perrin, Salk Institute, San Diego, CA, USA) as a template. A BglII restriction site (bold) was introduced into the forward primer (5¢-CCTGTCAGATCTCCGCCAT GGCTAACAATGCATCTCT-3¢), and a BamHI site (bold) was introduced into the reverse primer (5¢-TCTCC CGGATCCAAAGAGAAATACCCATA-TA-3¢) to facilitate vector–insert ligation. Ampli?cation conditions were 4 min at 92 C, followed by 35 cycles of 1 min at 92 C, 30 s at 55 C, and 2 min and 30 s at 72 C. A ?nal extension was carried out at 72 C for 10 min. PCR products were puri?ed by QIAquick PCR puri?cation columns (Qiagen, Hilden, Germany), and a double digestion with BglII and BamHI restriction enzymes was carried out. The PVL1393 BioGreen vector was linearized by BamHI digestion, and the prepared GnRH-R insert was ligated into the prepared vector. The ligation mixture was transformed into XL1-Blue cells (Stratagene, San Diego, CA, USA) according to the manufacturer’s protocol. A mouse GnRH-R/enhanced yellow ?uorescent protein (EYFP) expression plasmid was made by removal and replacement of GFP from the GnRH-R–GFP expression plasmid with EYFP. GFP was removed by BamHI and EcoRI digestions. The EYFP insert was synthesized by PCR using Pfu DNA polymerase and pEYFP-N1 vector (Clontech Laboratories, Palo Alto, CA, USA) as a template. PCR was carried out as described above using the forward primer (5¢-AATTCTGCAGTCGACGGT AC-3¢) and the reverse primer (5¢-GATTATGAATTCG AGTCGCGGCCGCTTTACTT-3¢). An EcoRI site (bold) was introduced into the reverse primer. The PCR product FEBS 2003 was puri?ed, and a double digestion with BamHI and EcoRI carried out. The GnRH-R–PVL1393 vector arm was prepared by removal of GFP from the BamHI and EcoRI sites, and the prepared EYFP insert was ligated into the vector arm. The ligation mixture was transformed into XL1-Blue cells according to the manufacturer’s protocol. PVL1393 BioGreen expression plasmid was used for the expression of cytosolic GFP. Cytosolic EYFP expression plasmid was constructed by the removal and replacement of GFP from the PVL1393 BioGreen vector with EYFP. GFP was removed from the PVL1393 BioGreen vector by BamHI and EcoRI digestions. The EYFP insert was prepared as described above, then ligated in the prepared vector. BIOL 475 Biological Imaging GFP and eYFP Paper Analysis The ligation mixture was transformed into XL1Blue cells according to the manufacturer’s protocol. Transfection and amplification of recombinant baculoviruses BTI Tn5 B1-4 cells (generous gift of S. Ford, Australian National University, Canberra, Australia) were used for expression of fusion proteins. BTI Tn5 B1-4 cells (Tn5 cells) are a cell line derived from the Trichoplusia ni egg cells and are commonly used for the expression of proteins using recombinant baculoviruses. Tn5 cells (0.7 · 106) were seeded in a T25 tissue culture ?ask containing 5 mL of Ex-Cell 405 medium (JRH Biosciences, Lenexa, KS, USA). The sample was placed at room temperature and the cells were allowed to attach ?rmly to ?ask (approximately 15 min). Transfection was performed using Lipofectin reagent (Life Technologies, Gaithersburg, MD, USA). The expression plasmids were cotransfected with the BaculoGold baculovirus DNA (Pharmingen), according to the manufacturer’s instructions. The transfected cells were incubated at 27 C for 4 days. Afterwards, culture medium was collected and used to infect freshly prepared cells for viral ampli?cation. An end-point titration was carried out to isolate a single clone. The recombinant baculovirus was ampli?ed to obtain a high titer stock solution by infecting freshly seeded Tn5 cells at MOI ¼ 0.5 UÆcell)1. The infected cells were incubated at 27 C for 4 days before the medium was harvested. Endpoint dilution was used to determine the viral titer. Baculovirus-based FRET assay (Eur. J. Biochem. 270) 4975 ent, Santa Clara, CA, USA) with the laser line set at 364 nm, and the ?uorescence was detected at an emission window of 480–602 nm. For the detection of GnRH-R–EYFP ?uorescence, the cells were illuminated by an Ar-visible laser (JDS Uniphase, San Jose, CA, USA) with the laser line set at 514 nm, and the ?uorescence was detected at an emission window of 520–602 nm. The cells were illuminated with minimum level of laser power, and images were recorded at a frame-average of eight. To minimize photobleaching and cell movement during imaging, the recording was completed in approximately 5 s. Spectral characterization of GFP and EYFP For GFP, GnRH-R–GFP expressing cells were illuminated with an Ar-UV laser and the laser line set at 364 nm. Spectral scanning was carried with the interval of scanning set at 2.24 nm. For EYFP, GnRH-R-EYFP expressing cells were illuminated with an Ar-visible laser and the laser line set at 488 nm. Spectral scanning was carried out as above. FRET assay Cell culture and expression of the GnRH-R–GFP, GnRHR–EYFP, cytosolic GFP and EYFP were performed as described above. The principle of the FRET assay is illustrated in Fig. 1. GnRH agonist (pGlu-His-Trp-Ser-TyrD-Ala-N-methyl-Leu-Arg-Pro-Gly-NH2; Sigma, St. Louis, MO, USA) was added to the culture at a ?nal concentration of 100 nM [37]. Five minutes after the addition of the GnRH agonist, the prepared cells were visualized by illumination with an Ar-UV laser and the laser line set at 364 nm. The GnRH-R–GFP expressing cells were observed in the green channel with the detection window at 484–512 nm. FRET, GFP ?uorescence bleed-through and EYFP emission resulting from the Ar-UV excitation were detected at the FRET channel with the detection window at 530–570 nm. The net FRET image was obtained after subtracting the GFP ?uorescence bleed-through and the emission of EYFP from Ar-UV laser excitation. GnRH-R–GFP and GnRH-R–EYFP expression To examine the expression and subcellular localization of the GnRH-R–GFP and GnRH-R–EYFP fusion proteins, protein expression using recombinant baculovirus was carried out by infecting freshly seeded Tn5 cells in a Laboratory-Tek II chambered coverglass (Nalge Nunc International, Naperville, IL, USA). The cells were infected with GnRH-R–GFP or GnRH-R–EYFP recombinant baculovirus at 3 MOIÆcell)1 and incubated at 27 C for 2 days. Cells expressing the recombinant proteins were detected with a Leica TCS-SPII confocal system (Leica, Heidelberg, Germany) ?tted to a DMIRBE microscope (Leica) using a 63 · 1.2 numerical aperture water immersion objective. The pinhole was set at 1 Airy disc unit, and an appropriate dichroic beam-splitting mirror was used. GnRH-R–GFP expressing cells were visualized by illumination using a Coherent Enterprise 651 Ar-UV laser (Coher- Fig. 1. Schematic illustrations of baculovirus-based ?uorescence resonance energy transfer (Bv-FRET) assay. GFP is fused at the C-terminal end of the mouse GnRH-R as a donor ?uorophore, and EYFP is fused at the C-terminal end as an acceptor ?uorophore and these fusion proteins are coexpressed in a cell line (Tn5 cells). GFP is excited by an Ar-UV laser at 364 nm, and energy transfer occurs from GFP to EYFP that emits yellow ?uorescence. The ?uorescence is detected at the FRET channel with the detection window at 530–570 nm using a Leica TSC-SPII confocal microscope. FEBS 2003 4976 T. C. Cheung and J. P. Hearn (Eur. J. Biochem. 270) The amount of GFP ?uorescence bleed-through subtracted was derived from the ?uorescence obtained from cells only expressing GFP. To ensure the GFP ?uorescence bleed-through from the GFP and EYFP coexpressing cells was fully subtracted, cells expressing GFP only should have similar, preferably equal, levels of GFP expression compared to that in the GFP and EYFP coexpressing cells. Similarly, the value of the EYFP background ?uorescence (resulting from the Ar-UV laser excitation) subtracted was based on the ?uorescence obtained from cells only expressing EYFP. To ensure this background ?uorescence from the GFP and EYFP coexpressing cell was fully subtracted, the cells only expressing EYFP should have similar, preferably equal, levels of EYFP expression compared that in the GFP and EYFP coexpressing cells. The subtraction (below) was carried out using the Leica TCS-SPII data analysis software (version 2002). FRET ¼ Total emission collection at 530570nm window GFP flourescence bleed-through EYFP emission resulting from the Ar-UV laser excitation: In addition, GnRH-R–EYFP expressing cells were visualized by illumination using an Ar-visible laser with the laser line set at 514 nm. Although this excitation wavelength was suboptimal for EYPE, it did not cause coexcitation of GFP. EYFP expressing cells were detected in the yellow channel with the detection window at 520–602 nm. Images were recorded at a frame-average of eight. Each experiment was repeated a minimum of three times. For the time series experiment, the yellow to green ?uorescence ratio as an indicator of FRET was measured in the presence or absence of GnRH analogues. Cell culture, protein expression, excitation setting and emission channels were the same as described above. A GnRH agonist (pGluHis-Trp-Ser-Tyr-D-Ala-N-methyl-Leu-Arg-Pro-Gly-NH2; Sigma) or antagonist (pGlu-D-Phe-Trp-Ser-Tyr-D-Ala-LeuArg-Pro-Gly-NH2; Sigma) was added to the cells at a ?nal concentration of 100 nM. Five minutes after the addition of GnRH agonist or antagonist, images from the green and FRET channels were recorded at a frame average of eight every 2 min for up to 20 min. A minimum level of laser power and duration of recording time were set for imaging to minimize photobleaching and cell movement during recording, respectively. The average intensity of the yellow and green ?uorescence in the membrane region was measured at each time point, and values were normalized to unity with reference to the set value at time zero. The yellow to green ratio was calculated and the values were plotted against time. Each assay was repeated at least three times. Results Receptor expression and subcellular localization Recent studies have shown that GnRH-R is capable of forming stable oligomeric complexes in … Get a 10 % discount on an order above $ 100 Use the following coupon code : NURSING10

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