Technical Reference #1611

1611.

A Novel Far-red Bimolecular Fluorescence Complementaion System that Allows for Efficient Visualization of Protein Interactions Under Physiological Conditions Jun Chu; Zhihong Zhang; Ying Zheng; Jie Yang; Lingsong Qin; Jinling Lu; Zhen-Li Huang; Shaoqun Zeng; Qingming Luo, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Biosensors and Bioelectronics, 25(1611), (2009)

Abstract:
Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells and bimolecularfluorescence complementation (BiFC) represents one of the newly developed imaging technologiesto directly visualize protein–protein interact

Keywords:
protein-protein interactions; bimolecular fluorescence complementation (BiFC); Far-red fluorescent protein; mKate; mulitcolor

Materials & Methods:
2.1. Expression constructs The following eukaryotic expression vectors were used in this study (for the detailed construction of the plasmids see Methods of Supporting Information). (i) Plasmids encoding fusion proteins of bFos (or bFos) and bJun with fragments of red FPs include pbJun-RN pbFos-RC and pbFos-RC wherein RN and RC represent the N- and C-terminal fragments of red FPs respectively. (ii) Plasmids encoding fusion proteins of EGFR signaling proteins with fragments of different FPs include pEGFR-LC151 pEGFR(C)-LC151 pSTAT5A-KN151 pSTAT5B-KN151 pEGFR-VN173 pGrb2-VC155 pbJun-CrN173 pbFos-CrC155 and pLC151-STAT5B wherein K L V and Cr stand for mKate mLumin (mKate-S158A) mVenus and mCerulean respectively. 2.2. Site-directed mutagenesis Site-directed mutagenesis was performed by the megaprimer PCR method using pRSETB-mKate as the template. The following oligonucleotide primers were used to make the mutations: mKate forward 5 -AAAAGGATCCGAATTCATGGTGTCTAAGGGCGAAGAGCTG- 3 mKate reverse 5 - TTTTCTCGAGATTAAGTTTGTGCCCCAGTTTGCTAGGGAGGTCGCAG- 3 S158X 5 -CACGAGCTTCAGGGCCATGTCMNNTCTGCCTTCCAGGCCGCCGT- 3 (mutated positions are underlined M stands for A or C and N for A T G or C). The PCR product was cut with EcoRI and XhoI cloned into the pRSETB vector electro-transformed into DH5 competent cells and then cultured on LB medium plates containing 50 g/ml ampicillin. After overnight growth at 37 ◦C the overall brightness of individual colonies was assessed using a fluorescence stereomicroscope (Leica MZ FL III;Wetzlar Germany) equipped with a DsRed filter (excitation 546/12 nm emission 620/60 nm) and 20 of the brightest colonies that were isolated were picked out and cultured in LB. Plasmids were extracted using a mini-prep kit further transformed into BL21 (DE3) and reexamined by fluorescence microscopy. The eight brightest colonieswere selected for sequencing analysis. 2.3. Protein expression and characterization mKate mutants were expressed from pRSETB vectors in Escherichia coli BL21 purified and characterized spectroscopically and biochemically as described previously with minor modifications (Jach et al. 2006; Shaner et al. 2004; Shcherbo et al. 2007). For detailed procedures see Methods in Supporting Information. 2.4. Cell culture and transfection COS-7 and HeLa cells were maintained in a humidified incubator at 37 ◦C with 5% CO2 and grown in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% newborn calf serum (NCS) 100 U/ml penicillin and 100mg/ml streptomycin. The day before transfection cells were seeded on 35-mm-diameter coverglassbottomdishes (MatTek Corporation Ashland MA) or 96-well plates and grown to 80–90% confluence. Transfectionswere carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. For ratio analysis plasmids encoding bJun and bFos (or bFos) fusion protein along with mCeruleanwere cotransfected in a ratio of 2.5:2.5:1 (0.075 g of pbJun-RN 0.075 g of pbFos-RC or pbFos-RC and 0.03 g of pmCerulean-C1) wherein mCerulean serves as an internal control to measure the BiFC efficiency. For EGFR and STAT5 interaction studies 0.075 g of pEGFR-LC151 or pEGFR(C)-LC151 was cotransfected with 0.075 g of pSTAT5-KN151. For multicolor BiFC assay equal amounts of cDNA for EGFR-VN173 Grb2-VC155 STAT5B-KN151 and LC151-STAT5B were used to have a total of 0.24 g cDNA while the amount of pbFos-CrC155 and pbJun- CrN173 was 0.025 g. After transfection the cells were incubated at 37 ◦C until imaging. 2.5. Fluorescence microscopy and image processing Confocal laser scanning microscopy was carried out with a FV1000 (Olympus Japan) controlled by the software FluoviewVer. 1.5. The confocal microscope was equipped with a HeNe-G laser using the 60× oil-immersion objective lens (NA 1.35). The BiFC mLumin fluorescence was excited at 543 nmand detected within a range from 580nm to 680 nm. Epifluorescence was performed on an inverted wide-field fluorescence microscope (IX71; Olympus Japan) equipped with a digital CCD camera (Qimaging Canada). An UPlanSApo 100×/1.40 oil objective (Olympus) and the following filter sets (Olympus) were used to obtain the fluorescence images—(1) CFP: excitation 425-445HQ dichroic DM450 emission 460-510HQ; (2) YFP: excitation 490–500 dichroic DM505 emission 515–560; (3) RFP: excitation 545–580 dichroic DM600 emission 610IF. All fluorescence images were corrected by subtracting the background and analyzed using ImageJ (http://rsbweb.nih.gov/ij/) and Adobe Photoshop 9.0. The ratio value representative of fluorescence complementation efficiency was calculated as IBiFC/ICFP wherein I represents fluorescence intensity.

Microscopic Technique
Fluorescence Microscopy, Confocal Laser scanning Microscopy

Cell Type(s)
COS-7, HeLa Cells