GFP Mutants with Altered Mass Spectra - Research Proposal Example

GFP mutants with altered mass spectra and fluorescence intensities Khalid Alquthami Leeds Several molecular biology techniques were utilized to purify, amplify and express both wild-type and mutant GFP proteins from cloning and expression vectors transformed into E. coli. Site-directed mutagenesis using specific primers was performed on GFPuv which was excised out of plasmid pET23. The mutant protein was expressed in E. coli using auto-induction technique. The mutant produced had lower mass-charge ratio, lower peak and maximum emission spectra compared to the wild-type. This shows that the mutation changed the chromophore properties. Keywords: GFP; EGFP; mutants; mass spectra; fluorescence 1. Introduction The green fluorescent protein (GFP), was discovered and isolated from thefrom the jellyfish Aequorea victoria (1). The native protein contains 238 amino acids which folds into six alpha helices and eleven beta strands forming a classical cylindrical beta sheet with anti-parallel strands. The GFP protein owes its fluorescence to the ser-tyr-gly derived chromophore p-hydroxybenzylideneimidazolinonev (2) that is buried deep in the the beta barrel which protects interactions with solvent. Due to its stable fluorescence, GFP has been used in biological studies as an important marker for processes like protein trafficking. Sequence data for GFP has led to the development of recombinant GFP proteins with modifications to increase the fluorescence intensity and longevity. There are many mutant GFPs that with differences in absorption and emission spectra. There are enhanced GFP (EGFP) that has higher-intensity emission after blue-light excitation compared to wild-type GFP (4) (3). Another is GFPuv, a recombinant GFP with maximum maximum emission spectra is 509nm, similar to that of wild-type GFP. GFPuv has a molecular weight of 29 kDA with three amino acid substitutions (Phe-99 to Ser, Met-153 to Thr, and Val-163 to Ala resulting in 18 times more brightness under ultra-violet (UV) light at 395nm. GFPuv is 18 times brighter than wild-type GFP. The large fluorescenc of GFPuv making it suitable for experiments that require detection of changes in fluorescence. GFPuv expressed in highly expressed in E. coli is a soluble, fluorescent protein in contrast to wild-type GFP, which may be expressed in inclusion bodies as a nonfluorescent protein. The aims of this study are to introduce mutations by site directed mutagenesis to recombinant GFPuv DNA sequence, express the mutated protein and compare the mass spectrometric properties and fluorescence intensity of the mutant GFPuv with the wild type GFPuv. 2. Materials and methods 2.1 Preparation of GFPuv for insertion Plasmid pET23GFPuv (5 µg) was digested with 30 units each of restriction enzymes Nde1 and HindIII in 5 µl of 10x restriction buffer in a 37oC water bath for a minimum of 3 hours. The digests were electropohresed on agarose gel. The resulting 761 bp DNA product was excised and purified using te QIAquik gel extraction kit (Qiagen). The purified DNA concentration was determined by measuring its abosorbance at 260 nm. 2.2 Cloning of GFPuv into E.coli The purified GFPuv was ligated to vector pET28c which has been pre-digested wth NdeI and HindIII, and purified using the QIAquick kit. The molar ratio of vector of GFPuv DNA to pET28c used was approximately 8:1. The ligation reaction also had 2 µl of 5x T4DNa ligae buffer, water and 1µl of T4DNa ligase. After an overnight incubation at in a 16oC water bath, 5 µl of the ligation reaction was added to 50 μl of DH5α competent cells. This was mixed by gently by pipetting up and down three times. Negative and sterile controls were also prepared. Tubes were then incubated on ice for 30 min and transferred to a 42oC water bath for exactly 20 sec after which the tubes were returned to ice for another 2 min. Then, 950 µl of SOC media pre-warmed to 37oC was added to each tube and placed at 37 oC. After 1 hour, 50 μl- 200 μl of transformation reactions were spread on LB agar plates containing 50μg/ml kanamycin. Plates were incubated overnight at 37oC then stored at 4oC. Colony PCR to verify successful insertion was performed by picking cells from well separated colonies and resuspending cells in sterile water and centrifugation. Twenty μl of the supernatant from each tube was placed in a 0.2 ml PCR tube with 0.2 mM dNTPs, 1μM T7 promoter primer, 1 μM T7 terminator primer, 1.5 mM MgCl2, 1x Taq polymerase buffer. The total PCR volume was 50 µl. PCR cycling parameters were as follows: 35 cycles of denaturation at 1 min, 94oC; annealing at 1min, 55oC, extension, 1 min, 72oC; followed by final extension for 10 min at 72oC. 2.3 Site-directed mutagenesis Site-directed mutaenesis was carried out using Quik-Change® (Stratagene) but used a different DNA polymerase. The reaction mixture was: 5 μl 10xPCR buffer for hot start KOD polymerase,5 μl 2 mM dNTP mix, 10-20ng GFPuv-pET28c template DNA, 125 ng M2F primer (5’-CACTTGTCACTACTCTCACTTATGGTGTTCAATGCTTTTCCCG-3’), 125 ng M2R primer (5’-CGGGAAAAGCATTGAACACCATAAGTGAGAGTAGTGACAAGTG-3’), 2 μl 25 mM MgSO4 sterile water to 49 μl and 1μl KOD hot start polymerase (1U/μl). Thermal cycling paramaters were as follows: initial denaturation at 94oC, 30sec followed by 24 cycles of denaturation 94oC, 30 sec; annealing 55oC, 1 min and extension 68oC, 4 min 20 sec. Final extension was 68oC, 10 min. PCR reactions were then digested with 1μl (10U) of Dpn1 for 37oC for 60 min. 2.4 Identification of mutations within GFPuv insert One μl of the DpnI digest was added to 50 μl of XL1 blue supercompetent cells. The reaction was placed on ice for 30 min then heat pulsed at 42oC at 45 seconds then immediately placed on ice for 2 min. After addition of 0.5 ml NYZ broth, transformations were incubated for 1 h at 37oC in a shaking water bath. Cells were spread to LB-kanamycin plates and placed at 37oC overnight. Single colony was cultured for small scale plasmid DNA purification. Three ml of E. Coli culture was centrifuged and pelleted. Resultant pellet made t undergo a series of washings with buffers P1and P2. Final resuspension was n buffer N3; after centrifugation the supernatant was applied to a QIAprep spin column. After several washes, the bound plasmid DNA was eluted with 50μl buffer EB. Five µl of the eluate was digested with 10U of Hind III for 1 h at 37oC. The digestion products were loaded to agarose gel for concentration determination. The GFP DNA fragment was excised from the gel and purified. DNA samples were also sent for sequencing. 2.5 Transformation of GFP mutants into Expression Host Competent cells of BL21(DE3)pLysS, an expression host for the pET vectors, were prepared from an overnight culture of the bacteria using standard protocols. Bacteria were added to SOB media containing 34μg/ml chloramphenicol and allowed to grow at 37°C until OD600 is >0.2 and Read More