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Wild type GFP and eGFP (Mutant) are cloned (pET28c expression vector) and expressed using site directed mutagenesis and was transformed to expression host E.Coli BL21 (DE3) pLysS. Protein is then expressed by auto induction method, isolated by western blotting, purified using Ni-TA chromatography and analysed by mass spectrometry and spectral analysis. The mutant GFP has a molecular weight of 29556 Da and the wild type GFP had a molecular weight of 29560 Da. The emission peak of the mutant is found to be 509nm and an intensity of 32.837 AU. Whereas the emission peak of wild type is found to be 504nm and had an intensity of 16.949AU.
KEY WORDS: Site Directed Mutagenesis, Ni-TA Chromatography, Mass Spectrometry, Fluorimetry.
GFP (Green Fluorescent Protein) is one of the major tools in the fields of molecular biology and cell biology. This magnificent protein is discovered by Shimomura et al from a jelly fish called Aequorea Victoria . The attractive feature of this protein is its ability to generate strong visible fluorescence without any additional cofactors. This protein contains 238 amino acids [1, 2]. The fluorescent chromophore p-hydroxybenzylideneimidazolinone is formed spontaneously by the residues 65-67which are Ser-Tyr-Gly . GFP has an eleven stranded Î²-barrel crystal structure which is threaded by Î±- helix, which runs along the axis of the cylinder. The chromophore is called as a Î²-can as it is attached to Î±- helix and is situated in the centre of the cylinder.
There are seven types of GFP variants . Class I is a wild type mixture of neutral phenol and anionic phenolate, Class II is a phenolate anion, Class III is a neutral phenol, Class IV is a phenolate anion with stacked Ï€-electron system, Class V is an indole, Class VI is an imidazole and Class VII is phenyl. These divisions are made based upon the components of their chromophores and spectral characteristics.
The wild type GFP has a major absorption at 398nm and minor absorption at 475nm [3, 4]. A maximum emission of 508nm is seen when the protein is excited at 398nm. When an irradiation took place at 475nm a maximum emission will be produced at 503nm. In wild type GFP there are two excitation peaks one at 395nm and the other at 475nm. GFP has various applications when compared to any other fluorescent proteins. An internal post translational autocatalytic cyclization produces the chromophore. This doesn't require any cofactors or substrates. Apart from these one of the major characteristics of this protein is its ability of resistance to heat, pH, detergents, photo bleaching, organic salts and many proteases .
Apart from its benefits this Green Fluorescent Protein has a few limitations . The posttranslational chromophore formation in this protein is very slow. It is hard to differentiate a densely localised or less expressed from the back ground fluorescence. One of its major drawbacks is its requirement of oxygen.
The present work is to create mutations in the wild type and Mutant and to compare their characteristics and spectral properties. In the mutant the mutations takes place at 64th position which is phenylalanine replaced by Leucine (F64L) and at 65th position which is serine will be replaced by threonine (S65T).
MATERIALS AND METHODS:
All chemicals, reagents, cells, etc are purchased from NOVAGEN and protocols followed are from NOVAGEN pET system manual, 11th edition.
Measuring the Concentration of DNA Solution and Restricted Digestion:
The plasmid DNA pET23GFPuv is quantified by using the spectrometric methods at two different wavelengths A260 and A280 at a dilution rate of 1:100 and the ratio of these two wavelengths are measured. The 761bp in length GFPuv open reading frame located in between the sites Hind III and Nde I of pET23GFPuv is subjected to restriction digestion using 30 units of both Hind III and Nde I enzymes. After performing the restriction digest it was analysed by performing 1% agarose gel electrophoresis along with 1kb DNA ladder. The 761bp length containing the GFP is excised from the gel and the DNA is extracted from the gel by using the QIA quick gel extraction kit from Qiagen using the standard protocol provided along with it.
Ligation Reaction and Transformation of E.Coli:
The pET28c which is 5367bp in length (digested with Nde I and Hind III enzymes) along with the 761bp in length GFP are left for ligation reaction with an 8:1 insert to vector molar ratio using the T4 DNA ligase and was left over night at 16oC for incubation. After the incubation 5Âµl of the ligated pET28c-GFP is transformed into E.Coli DH5Î± cells at 42oC for 20seconds (Heat Shock Method). These cells were then transformed into agar plates containing 50Âµg/ml of kanamycin and incubated overnight at 37oC. A colony PCR is performed one a single colony in order to find out the plates contain the pET28c plasmid along with GFP inserted. The PCR reaction contains a mixture of 0.2mM dNTPs, 1ÂµM of T7 promoter primer and T7 terminator primer (both 5 pmol/Âµl), 1.5mM MgCl2 and 1x Taq polymerase buffer. 1 cycle of PCR performed at 1min 94oC, 1 min 55oC, and 1 min 72oC. This is performed for 35 cycles followed by 10 min final extension at 72oC. The PCR products are then analysed by using 1% agarose gel electrophoresis with 100bp DNA ladder.
Site Directed Mutagenesis and Transformation into XL-1 Super Competent Cells:
Site directed mutagenesis is performed by using Quick-ChangeÂ® site directed mutagenesis kit protocol marketed by Stratagene, but using 1Âµl KOD hot start polymerase (1U/Âµl). The primers used for these are
Forward: 5'-CACTTGTCACTACTCTCACTTATGGTGTTCAATGCTTTTCCCG-3' Reverse: 5'-CGGGAAAAGCATTGAACACCATAAGTGAGAGTAGTGACAAGTG-3'
The samples are then incubated in PCR at 94oC for 30sec, 24 cycles at 94oC for 30sec, 55oC for 1 min, and 68oC for 4min 20sec. A final incubation is done at 68oC for 10 min. 1Âµ1 (10U) of Dpn1 is added to the reaction and was incubated at 37oC for about one hour. 1Âµ1 of Dpn1 digest is added to the super competent cells. The cells are then subjected to heat shock at 45seconds at 42oC and left in ice for 2 min. 0.5ml of NZY+ broth is added and incubated at 37oC for one hour in a shaking water bath. The transformation reaction is then transformed into LB agar plates containing 50 Âµ1 of kanamycin and left overnight for incubation at 37oC.
Extraction of Plasmid DNA from Transformation colonies and Sequencing:
By using the QIAprep Miniprep kit from Qiagen the plasmid DNA is collected from the transformed colonies and was analysed by agarose gel electrophoresis in order to check the purity and concentration of the extracted DNA. The DNA was then sent to Integrated Genomics and Gene Expression Analysis Facility, Faculty of Biological Sciences, University of Leeds.
Transformation into Expression Host E.Coli BL21 (DE3) pLysS and auto induction:
1 Âµl of Plasmid DNA is added to the competent cells and was subjected to heat shock treatment (in ice for 2 min, 42oC in water bath for 30 sec and in ice for 2 min). These cells are then transformed into the LB Kan/Cam plates and left overnight for incubation at 37oC. In order to perform the auto induction some of the culture is transferred to the SB-5052+ Kanamycin media, which is best for auto induction process.
Simple Cell Fractionation and SDS PAGE Analysis:
In order to fractionate the soluble and insoluble cells 2ml of Bugbuster reagent and 1Âµl of DNAase is added to the total induced sample. The cells are then separated by centrifugation at 13,000rpm for 20 minutes. The fractionated cells are then analysed by using SDS PAGE. The fragment is then analysed by using a low molecular weight ladder and then transferred to coomassie blue stain.
Western Blotting and Chemiluminescent Detection:
Western Blotting is performed by using Nitrocellulose membrane (Blotting paper) and by using Biorad 3000 series power pack. The protein then gets transferred into the nitrocellulose paper by running the Biorad 3000 series power pack continuously for one hour at 250mA using ponceau S solution. After running the blot the ponceau S solution is completely drained and the blot paper is then transferred to the His probe HRP working solution (diluted stock solution in 1:5000 of blocking buffer). The blot is then incubated in the solution for 45 minutes at room temperature with shaking. Then the blot is developed by using a chemiluminescent detection kit and then in X-O-Graph machine.
Ni-TA Chromatograph and Bradford Essay:
His bind resin slurry is centrifuged at 3000rpm for 1 minute. The Ni resin present in the slurry is equilibrated by washing with 800Âµl sterile deionised water (2 washes), 800 Âµl 1x charge buffer (50mM NiSO4 3 washes) and final wash with the elusion buffer (0.5M NaCl, 20mM Tris HCl, 5mM imidazole and pH 7.9). This makes the soluble fraction. The unbound sample is prepared by adding 10X binding buffer to the soluble fraction and centrifugation at 3000 rpm for 1min. The resin is washed for 2 times with 12000 Âµl 1x wash buffer and was centrifuged at 3000rpm for 1 min. This produces the washes 1 and 2. The bound protein is then eluted with 800 Âµl of 1x elusion buffer for 2 times in order to produce the elutions 1&2. 20 Âµl of each fraction (total soluble fraction, unbound, wash1, wash2, Elution1 and Elution 2) are then subjected to SDS PAGE analysis using 40-20% of agarose gel. The purity of the sample is then detected by using SDS PAGE analysis along with Coomassie Blue staining. The protein concentration is then measured by using the Bradford Essay technique.
Mass Spectrometry and Fluorimetry:
The protein is then analysed by using the Electron Spray Mass Spectrometry. The samples are then treated with 15 Âµl of methanol and 1% formic acid. This solution is then transferred into a standard Platform II electron spray ionisation source (Waters UK Ltd, Manchester, UK) through a stainless steel capillary at a flow rate of 4-1000 Âµl /min and was operated at a high voltage of 4KV. Nitrogen which is used as both nebulising gas and drying gas (warm gas) flows on the outer surface of the capillary. Data is acquired using mass to charge analysis for a 10 second scan. The fluorometric analysis is performed at an excitation wavelength of 450nm, accumulation 5, emission range 490nm-550nm and slit width 4 and 4.
The DNA sample concentration was found to be 0.036mg/ml and A260/A280 ratio is found to be 2.028 which indicate the purity of the given DNA. 761bp in length GFPuv is excised and extracted from the agarose gel. GFP ORF is ligated with pET28c is transformed into E.Coli DH5Î± cells and positive clones are picked by using the colony PCR method. The required mutations S65T, F64L is created within the GFPuv insert that is cloned into pET28c through site directed mutagenesis method. The plasmid is then transformed into XL-1 Super competent cells. In the site directed mutagenesis a change was seen at 64 and 65 positions of amino acids (Fig 1). Positive clones are picked from LB agar plates containing kanamycin. The plasmid DNA is extracted and concentration of plasmid DNA was found to be good enough to carry out further process. The GFPuv carrying plasmid is then transferred into E.Coli cells (BL21 (DE3) pLysS). The positive clones are then identified and cultured.
Protein expression is induced in the cultured positive clones through auto induction method and was confirmed by running 1% agarose gel electrophoresis. Due to the presence of the T7 promoter there is an increase in protein concentration. The cells are then harvested and fractionated into soluble and insoluble fractions. By SDS PAGE analysis of the fractionated cells it was clearly found from the bands that there is an increase in protein concentration. GFP is detected by using the Western Blotting and Hisprobeâ„¢ -HRP (Fig 2). An increase in protein concentration of induced sample and large amount of protein in soluble fraction (GFP Mutant) is found.
The identified protein is then purified by using the Ni-TA chromatography (Fig 3). By SDS PAGE analysis it was found that, the presence of GFP is seen in major amounts in total soluble fraction and in Elution 1&2. A trace amount of GFP is also seen in wash 1&2. The protein samples are then analysed by using mass spectrometry and Fluorimetry. From mass spectrometry results it was found that the mutant GFP has a molecular weight of 29556 Da and the wild type GFP had a molecular weight of 29560 Da (Fig 4). From the Fluorimetry results the emission peak of the mutant is found to be 509n and an intensity of 32.837 AU. Whereas the emission peak of wild type is found to be 504nm and had an intensity of 16.949AU (Fig 5).
Site directed mutagenesis is performed after the transformation of wild type and mutant GFP into the expression host E.Coli cells (BL21 (DE3) pLysS) by using T7 promoter as controller. The desired mutations are S65T and F64L. A codon changes were seen at 190th position. In this there was a change from TTC CTC, which shows that phenyl alanine is replaced by Leucine. A similar change is seen in TCT ACT, which shows that serine is replaced by threonine. These changes are conformed based upon the codon table.
The plasmid is then transformed into the expression host and protein expression is induced through auto induction method. These cells are then fractionated to get soluble and insoluble fractions. The protein expression is then detected using SDS PAGE electrophoresis. From the gel images it was found that there is an increase in protein concentration. Thick bands are found in induced and soluble fraction.
This will be confirmed by the using His-probe where the reaction between the HRP and luminol is the major principle behind the chemiluminescent detection. From the His-BLOT image it was clearly evident that GFP is present in the gel and will be further purified by using Ni-TA chromatography. The resin which holds the GFP protein binds to the His tag and was collected by washing it with washing buffer and GFP is collected using elution buffer. This is done twice. The rest of the soluble, unbound and wash are collected and then analysed through SDS PAGE. From the gel image it was found that a major amount of GFP is present in soluble, Elution 1&2.
A trace amount of GFP is also found in wash1&2 which might be the result of resin saturation. The purified GFP is collected and was further sent to spectral and fluorometric analysis.
From mass spectrometry results it was found that the mutant GFP has a molecular weight of 29556 Da and the wild type GFP had a molecular weight of 29560 Da (Fig 4). From the Fluorimetry results the emission peak of the mutant is found to be 509n and an intensity of 32.837 AU, whereas the emission peak of wild type is found to be 504nm and had an intensity of 16.949AU (Fig 5).
Thus the desired mutation (F64L, S65T) has been successfully created.
Figure1: CLUSTAL W Multiple Sequence alignment showing the nucleotide change in 190 and 193 positions. These results in the change of amino acid change of S65T and F64L
1 2 3 4 5
Figure 2: His BLOT image showing the presence of GFP bands in the gel produced by using chemiluminescent detection method. Ladder (1), Uninduced (2), Induced (3), Insoluble (4) and Soluble (5).
L TS UB W1 W2 E1 E2
Figure 3: Ni-TA chromatography image of different fractions. Ladder (L), Total Soluble Fraction (TS), Unbound (UB), Wash1 (W1), Wash2 (W2), Elution1 (E1), Elution2 (E2).
Figure 4: Electron Spray Mass Spectrometry image of mutant protein produced using nitrogen as a nebulising agent and as drying agent (Warm nitrogen).
Figure 5: Fluorometric Image of emission peak of the mutant protein and wild type when excited at 450nm.