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Green Fluorescent Protein (GFP) discovered by Shimonura et al. (1) at present is used widely in the field of molecular, medical and cellular biological studies (tseinAP11). It has a wide application as a marker gene in studies of cell apoptosis, dynamics of cytoskeleton and inhibition of gene expression (AP12) and can be used in studies conducted in various species ranging from plant, fungal to mammalian cells (AP10). Isolation of the chemiluminescent protein is carried out from the Aequorea Jellyfish (AP7).
From the experimental studies carried out earlier related to structure of GFP, the protein of 238 amino acids contain an α-helix that runs through the 11 stranded β-barrel (24˚A diameter and 42˚A height) in which helps in protecting the chromophore that lies at the center (AP7,AP8,AP10). The chromophore responsible for the fluorescence property of GFP is a 4-(p-hydroxybenzylidene)imidazolidin-5 . Amino acid residues Ser-Tyr-Gly at 65-67 positions are responsible for the formation of chromophore(AP7) through an autocatalytic O2 dependant process (AP7,8). O2 is hence required for the maturation of GFP after which the requirement of O2 is not an essential factor.
The excitation peaks of GFP can be observed at a wavelength 395 nm which gives rise to a major peak and 475 nm which gives rise to a minor peak and emission at 503-308nm respectively to the excitation peaks. However, fluorescence property of GFP can be observed only when the β can structure is present. When this structure is denatured the fluorescence property is completely lost and regained when the structure is reformed hence the structural stability of GFP plays an important role in its function.
It has also been seen that formation of mature GFP is favourable only at temperatures below 37˚C, which limits the use of GFP. In order to overcome the limited use of wild type GFP, mutants have been created that have the capability of maturing at higher temperatures. The mutants also have an ability to reduce the localisation of proteins in inclusion bodies compared to that of wild type GFP who localise the proteins in inclusion bodies due to misfolding (AP10). The mutants that have been created generally have a higher fluorescence intensity compared to that of wild type (AP3CUbittetal) and have an ability to sustain a high pH range when compared to wild type GFP which loses its fluorescence capacity at pH 11-12.
Yellow Fluorescence protein (YFP) was the initially designed Green Fluorescent mutant creating a mutation at the T203Y position. During the late 90s blue, green and cyan fluorescent proteins were developed. Different fluorescence spectrums of these mutants were obtained due to the difference in the imidazolinone based chromophore and the chromophore interaction with protein environment (AP8).
Depending upon the chromophore component seven classes of GFP mutants are derived. Classes 1-4 chromophores, obtained from polypeptides with Tyr at 66th position and classes 5-7 obtained from Trp, His and Phe at the 66th position (AP7/8).
The main objective of the current study is directed towards creating the class 5 mutant Cyan Florescent Protein (CFP), one of the GFP mutants that has increased its use exponentially in recent years. Recent studies have showed that the fluorescence properties of CFP is widely used in combination with GFP and YFP in studies dealing with transgenic mice containing fluorescent neurons (AP8). CFP is widely used in FRET based applications in combination with YFP ().
The experimentation has been carried out by using site directed mutagenesis strategy to introduce a mutation at the 66th Tyrosine of the wild type GFP, converting to Tryptophan (Y66W) and transforming the mutated products into E.coli BL21(DE3) cells after which the proteins were expressed by the auto induction method. The presence of the proteins was verified by western blot, followed by purification carried out by Nickel Affinity Chromatography. The fluorescent results obtained by fluorometry of the mutants will be compared with that of GFP.
2. Materials and Methods
2.1 Construction of GFP cloned pET28C
PET23GFPuv plasmid (Novagen) was digested using 20U/µl NdeI and 10U/µl Hind III enzymes. Presence of GFP was analysed running a 1% agarose gel. The 761bp fragment was purified using QIA quick gel extraction kit as for Qiagen protocol. Purified fragment was ligated to pET28C vector (Novagen) which was pre digested by NdeI and Hind III enzymes, in presence of 5x T4 DNA ligase buffer and 1U/µl T4 DNA ligase and incubated overnight at 16˚C.
2.2 Transformation to E.Coli cells
Ligated products was transformed to 4xDH5α cells by heat shock method and plated in LB agar/ Kanamycin for identification of successfully transformed cells. Colony PCR was carried using 0.2µM T7 promoter and terminator primers, 0.2mM dNTPs, 1.5mM MgCl2, 1X Taq polymerase buffer. PCR of 35 cycles with 94˚C 1min, 55˚C 1 min, 72˚C 1min and final extension 72˚C 10min was carried out.
2.3 Site directed Mutagenesis (SDM)
SDM was performed using Quick- change® site directed mutagenesis kit (Stratagene). KOD hot start polymerase was used due to the high fidelity and faster elongation rate than can be obtained than the Pfu Turbo DNA polymerase provided in the kit. PCR reagents 10x PCR buffer for KOD start polymerase, 2mM dNTP, 10-20ng GFPuvpET28c template, forward primer 5'-CACTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCC-3' , reverse primer 5'-GGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTG-3' for CFP mutant , 25mM MgSO4, 1U/µl of KOD hot start polymerase were used. PCR conditions 94˚C 30seconds, 24 cycles of 94˚C 30seconds, 55˚C 1 minute, 64˚C 4minutes 20 seconds and extension of 68˚C 10 minutes was maintained. PCR products were digested using 10U Dpn I for digestion of dam methylated parental template.
2.4 Transformation to XLI supercompetent cells
Digestions were transformed to 2x XL-1 blue supercompetent cells by Stratagene through heat shock method followed by incubation in NZY+ broth at 37˚C 1hour and plated in LB Kanamycin media. Transformed DNA was extracted using QIA prep mini kit (Qiagen) following manufacturer's protocol. The DNA was quantified by nano drop technique and sequenced.
2.5 Expression and verification of CFP
Plasmids were transformed to T7 expression host E.coli BL21 (DE3) cells (Novagen) through heat shock method. Transformed cultures were plated on LB Kanamycin/ Chloramphinicol media and incubated over night at 37˚C. LB-1D media was inoculated by cultures and OD600 of was obtained which is the non-induced fraction. SB-5052 auto induction media and 100µg/ml of Kanamycin was used to induce protein expression. CFP was incubated at 28˚C 20hours and GFPuv at 37˚C 20hours at 350-400rpm resulting the induced sample. Pelleting followed by resuspending in SDS-PAGE buffer of induced and non-induced samples were carried and stored in ice. The cooled samples were fractionated using BUGBUSTER™ (Novagen) and 1µl of DNAse1. Supernatant obtained by centrifugation was used as soluble fraction and pellet suspended in 2ml Binding buffer was used as insoluble fraction. Presence of proteins was verified running a 12% SDS-PAGE and staining by Coomassie blue dye. Western Blot analysis of the proteins was carried out using Hisprobe™-HRP (Novagen). The blots were detected by Chemiluminescent detection kit which contained Luminol enhancer and Peroxide buffer and films were developed in an XO-graph machine.
2.6 Purification of Protein
To soluble fraction, 10x binding buffer was added along with His binding resin and was subjected to washes by sterile deionised water, 1x charge buffer (50mM NiSO4) and 1x binding buffer (0.5M NaCl, 20mM Tris HCl, 5mM imidazole) and centrifuged. The supernatant was labelled as unbound fraction. Resin was washed using 1x binding buffer followed by two successive washes by 1x wash buffer ( 0.5mM NaCl, 60mM imidazole, 20mM HCl, pH7.9) for washes 1 and 2 and 1x elution buffer (1M imidazole, 0.5 M NaCl, 20mM Tris HCl pH7.9) for elution 1 and 2. Purified DNA was run in 4-20% SDS-PAGE gel.
2.7 Analysis of GFP
The protein concentration of elution 1 was measured by Bradford assay, treated with methanol and 1% aqueous formic acid and infused to ESI-Mass Spectroscopy with 3.5kV for ASI capillary and Nitrogen gas as nebulising and drying gas.
Fluorometry was carried for spectral analysis with 10 µg/ml CFP parameters excitation 440nm, emission 460-550nm and 1 µg/ml GFP excitation 450nm, emission 490-550nm using slit width of 4 and 4 and accumulation 5 in case of both cases.
3.1 Sequencing analysis
The PCR products of transformed DH5α cells were subjected to side directed mutagenesis, followed by the sequencing. The results were translated to the corresponding amino acid sequence by using expasy and aligned using clustal W. According to the results of the sequencing data (Figure 1) obtained, in order to clarify the presence of the mutation, it can be seen that the mutation has successfully been created at the 66th position of the amino acid resulting in an amino acid conversion from tyrosine to tryptophan (Y-> W) which results in the CFP mutant production. It was also observed that 234th amino acid of the CFP has not been identified.
GFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60
CFP MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60
GFPuv VTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120
CFP VTTFSWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120
GFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180
CFP NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180
GFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238
CFP HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMXELYKT 239
Amino acid sequence alignment of GFPuv and CFP from sequence results obtained by SDM (The vector DNA sequence was edited and the sequences are considered from the NdeI site). Stars show the identical residues while double dots indicate difference in the amino acids. (Own data- 006)
3.2 Detection of the presence of CFP
The western blot analysis carried out by using the His Probe and detected by chemiluminescent method results in bands (Figure 2) between the marker range of 24-33 kDa in case of the induced, insoluble and soluble samples. The presence of the bands confirms the expression of CFP protein which has a molecular mass of around 27kDa.
Figure 2- Western blot image result obtained during the chemiluminescent detection of expressed CFP. Molecular marker (kDa) (Lane 1), Uninduced sample (Lane 2), Induced sample (Lane 3), Insoluble sample (Lane 4), Soluble sample (Lane 5). (Sample 15)
3.3 Purification of CFP protein
The protein samples clarified by western blot were purified by Nickle affinity chromatography followed by running in an SDS PAGE (Figure 3). The protein bands were obtained in the molecular weight range of 24 -33kDa which corresponds to the band length of the CFP. The band intensity of the total soluble fraction was high to that of the unbound fraction. The washes gave a slighter band intensity compared to that of elution.
Purified samples by Nickle affinity chromatography detected by running in a 12% SDS PAGE. Molecular marker (kDa) (Lane 1), Total soluble unbound sample (Lane 2), Bound sample (Lane 3), Wash 1 (Lane 4), Wash 2 (Lane 5), Elution 1 (lane 6), Elution 2 (lane 7).( Sample 19 c)
3.4 Mass spectroscopic analysis of GFPuv and CFP
The mass spectroscopic analysis (Figure 4a and 4b) of GFPuv resulted in an atomic mass of 29,570.75 Da where as the spectrum for the CFP resulted in a comparatively high mass of 29597 Da.
3.5 Fluorometric study of GFPuv and CFP
The results obtained from the flourometric spectrum show that the emission wavelength corresponding to the maximum peak of GFPuv is 502.5 nm where as the CFP emission wavelengthcorresponding to the maximum peak is 477nm. A difference in the shift of 25.5 nm can be observed between the two peaks of the emission wavelengths of the proteins. It is also to be noted that the intensity of fluorescence in case of the CFP protein has been lowered in 34 folds when compared to that of GFPuv. From the results that are obtained it is also to be seen that the CFP contains a wider range of emission wavelength when compared to that of GFPuv.
Figure 5- Fluorometric results obtained by excitation of GFP at 450nm and excitation of CFP at 440nm. ( Data from VLE)
The CFP had been successfully created using site directed mutagenesis strategy which is one of the best methods of producing mutants with respect to a single amino acid change in order to study the effect of substitution of the amino acid on the functional aspects of a protein (AP1) which in the current study is the replacement of Tyrosine by Tryptophan. The mutation results in producing an indole, instead of phenol or phenolate that is present in a GFPuv chromophore. Mutation carried out on GFPuv belongs to the class5 mutant category with respect to the classification carried out by Tsein on GFP mutants (AP7).
The CFP expressed by auto induction method in BL21 (DE3) cells were analysed by the western blot technique due to the presence of the His-tag that was present in the vector. The His-tag was detected by the use of Horse Raddish Peroxidase (HRP) enzyme which resulted in oxidation of luminol and produces chemiluminescent light that is detected by the XO graph. Due to the reason that all mutant proteins of GFP are soluble in nature () the intensity of the band seen in the soluble sample is shown to be high. Due to the presence of the of the His- tagged in CFP protein it was able to be purified by Ni-affinity chromatography which binds only proteins that are only His-tagged followed by running the SDS PAGE. The wash 1 and wash 2 contain slight bands due to the reason that the proteins can be washed off away during the purification process. However, the elutant 1 contains the highest intensity band due to which it can be used as purified samples. The elutant 1 was hence sent for mass spectroscopic analysis.
Upon analysis the mass spectroscopic data it can be seen that experimentally atomic mass obtained in case of CFP and GFP are the high when compared to the atomic mass that are already discovered in previous studies, which states that the atomic mass of GFPuv is of 26.8kDa() . This can be due to the presence of the His-tag that has been not been removed before the mass spectrometric analysis. It can also be observed that the mass of the CFP is comparatively high which can be due to the mutation that has been created.
According to the GFPuv data that have been obtained previously the spectrum results contain a major excitation peak at 395nm which emission peak is at 508nm wave length and a minor excitation peak at 475nm which emission peak wavelength is 503nm. Thus the emission peak depends upon the wavelength of excitation. The 475nm peak of the GFP is resulted due to the presence of the anionic or deprotonated chromophore where as the 395 minor peak is due to the presence of neutral or protonated chromophore(ZImmertsein). Howerver, the neutral protonated species become acidic upon excitation due to which they exist in deprotonated form after excitation(tsein) which results in chemically similar species to that generated by the 475nm excitation peak. Due to this reason, only one peak of distinct emission spectra can be seen corresponding to 502.5 nm which is very close to the theoretical emission hump at 503nm. According to the previous studies carried on CFP mutants, the excitation spectrum contains a peak at 436nm due to the intermediate between the neutral and anionic phenol and the emission peak at 476nm. The emission peak 477nm that is observed during the studies carried out is very much similar to that of the results that were expected. The emission wavelength corresponds in the production of blue-green / cyan fluorescence. It was also observed that the intensity of the CFP is 34 folds low that the GFPuv which results in low fluorescence. The low flurescent intensity of the CFP may be due to inefficient folding or due to the in efficiency of the chromophore formation(AP15). High emission of fluorescence in case of GFP can result in the damage of cells due to the production of free radicals which makes GFP unsuitable for long scale experiments which can be avoided by using CFP due to the low emission intensity (AP12). Efficient spectral line separation also can be obtained due to the reduced intensity of emission which can be used in expressing two fluorescent tags simultaneously (AP5) in conformational studies of different proteins located in various cell compartments in combination with other fusion tags.