IDENTIFICATION OF RECOMBINANT GFP PROTEIN USING CLONING, EXPRESSION AND PURIFICATION
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Course
BIO MISC
Institution
BIO MISC
IDENTIFICATION OF RECOMBINANT GFP PROTEIN USING CLONING, EXPRESSION
AND PURIFICATION
Abstract. 144
Green fluorescent protein is an useful tool to
observe the presence of protein-protein
interaction as well as gene expression(Chalfie
et al., 1994). Which those experiment include
amplificati...
1 IDENTIFICATION OF RECOMBINANT GFP PROTEIN USING CLONING, EXPRESSION AND PURIFICATION Khachonphat leesahatsawat 20301778 Abstract. 144 Green fluorescent protein is an useful tool to observe the presence of protein -protein interaction as well as gene expression (Chalfie et al., 1994) . Which those experim ent include amplification and recombination of GFP open reading fr om using RT -PCR which can clone GFP and insert i nto pQE3 0 vector with IPTG (Riedy et al., 1995) . which is used to promote expression of N terminal hexahistidine tag GFP , ampicillin resistance marker and hexahisti dine tag, then is purified by using IMAC immo bilized metal affinity chromatography which can detect the purity and yield of the GFP present from gel electrophoresis by using formul a of 260/280 from PCR, purity of GFP can be identify which should be in betw een 1.8 -2.0. Then observation of transformat ion of G FP in pQE30 can be analy ze by u sing agar pla te and can be seen und er UV transillumination. Which can be help in identification of GFP pQE30 growth and insertion of GFP in bacterial DNA. INTRODUCTION (300 words) . GFP or green fluorescent protei n is a protein with 238 amino acid residues which is extracted from jellyfish Aequorea Victoria that contain green fluoresc ence when exposed to light(Ormö et al., 1996) . It is being used as a marker protein to be able to observe a nd identify presence of particular protein s in the organic structure , there is also enhanced GFP which is a GFP mutant with b etter an d brighter fluorescence which make the detection easier and more sensitive, these technique have been used widely used for detection of gene transfer in mammalian cells. Such as integration of GFP ORF into pQE -
30 can be do ne by using molecular clon ing whic h transfer specific DNA sequence fr om donor to recipient vector such as hexahistidine -tag which will be used during this ex periment. RT-PCR or real -time polymerase chain reaction is a testing technique which can be used to amplify the GFP gene that is inse rts within BamHI and HindIII restri ction sites which presen ce in pQE -30 and therefore it enable the formation of complement ary sticky -ends to ensure optimal ligation (Miller et al., 2000) . Transformation of cells or bacteri a is then introduced , due to its i mportance in step for selection of bacteria (Chung et al., 1989) . Heat -
shock transf ormation is a techniq ue where the cells are exposed to sudden increase in temperat ure which will leads to ind uces the formation of pores and therefore the supercoiled plasmid DNA can enter the cell (Froger and Hall, 2007) . Purification of GFP by using IMAC will then be intr oduced , immobilize d metal affinity chromatogra phy is a purification technique which is used to detect interaction between proteins in solution and position of metal coordinating residues on the protein surface such as exposed histidine and cysteines presen t(Magnusdottir et al., 2009) . In this experiment, the aim is to clone GFP into pQE30 and identify the presence of hexahistidine -tagged recombinant GFP by using the help of purification of GFP usin g IMAC. EXPERIMENTAL PROCEDURES (800 word max) Amplification of GFP ORF using PCR and analysis by agarose electrop horesis , GFP ORF was a mplified b y adding 24.25 ul of steri le H20, 5ul of 10x vent buffer, 5ul pE -GFP, 5ul primer E1, 5ul primer E2, 2.5mM of dNTP, 0.75ul vent polymerase and was diluted in 150ul of sterile water and 5ul of 6x DNA sample buffer. DNA gel electrophoresis was per formed on 0. 8% w/v a garose gel with SY BR SAFE in 1x TBE buffer which performed at 120V for 30 -
45minutes. 2 Restriction Enzyme Digest ion and Purification of GFP and pQE -30 vector . 18ul of GFP and pQE30 DNA we re prepared follow up by 2ul of 10x buffer E and 5ul of BamHi/ HindIII enzyme premix , then incubation for 2 hours at 37 degree Celsius , Promega SV mini co lumns is then used to perform p urification of DNA. Transformation of recombinant pQE30 vector into competent E . coli cells . The GFP obtained was ligated into the pQ E30 by performing 3 tubes, one with both GFP and pQE30 DNA and tube 2,3 will contain both GFP DNA and pQE30 DNA respectively. Which 5ul of digested DNA will b e insert into tube 2 and 3 and ligated. 50ml of chilled cultured E. coli cell can be o btained by u sing ino ue methods, follow up by centri fugation at 4000rpm at 4 degree celcius for 10 minutes, discard the supernatant and add 1ml of Inoue transformation buffer and resuspend the cell pellet fo llow up by centrifu ge again to discard the super natant . 150ul of DMSO is then mix wi th the cell suspension and leave in ice bath for 10 minutes. Then is freeze in liquid N2 for transformation. Transformation result s can then be analyzed b y using plasmid mi ni prep and hexahistidine tagged GFP induction can be obtained . Trans formation of GFP -pQE30 into competent E. coli cells or XL -1 Blue E.coli cells. 3 samples w ere prepared, one is pQE30 GFP with vector and insert, one with vector only control and with in sert o nly control. Incubate of three sampl es at 65 degree Celsius for 5 minutes and incu bate at room temperature for 5 minutes. Heat-shock method is being used to trans formed into com petent cells by adding 0.5ml of Luria broth to each tubes and shake at 37 degree Celsius for 1 hour. I ncubation will occurs at 37 degre e Celsiu s and s tore at 4 d egree Celsius . Transformation result s can then be observe by observe by creating plate with positive GF P and negative GF P by inoculation cultures. Induction of synthes is of Hexahistidine -
tagged GFP in transformed cells. 1,5ml of each GFP positive and GFP negative was obtained and prepare for plasmid miniprep. GFP with TB wass inoculated into LB media with 25mg/ml ampicillin until OD600 reach 0.2 and incubate at 37 degre e Celsius and let it gro w for an hour with shaking until suffi cient de nsity reading o f 0.6-1.0 is o btained. Then plasmid miniprep can be prepared by using the manufacturer protocol SV minipreps DNA purification system. By adding 100mM of IPTG stock to give final con centration of 1mM and allow the cell to grow in incu bator , record the OD600 of the cell suspension every hour and transfer 1ml aliquot to fresh icro fuge tube and indicate +1 for every hours . After transfer the remainder of induced cells from fl ask to 50ml of scre w-capped plastic tube . Is then centrifuge at 4000rpm for 15 minutes and discard the supernatant . IMAC Purification of GFP. The pre -
measured 5ml of lysi s buffer are used and pour into E. coli cells with 2houurs post induction sample and in cubate the mixture for 20 minutes at 37 degree Celsius water b ath follow up by a dding 15ul of 1M MgCl2 and 20ul of 1mg/ml DNase to the mix ture and incubate at room temperature for 10 minutes . The lysate was sonicated for 10 seconds on ice and centrifu ge th e microfuge tube with 1ml of crude cell homogen ate for 1 minut e at full speed and discar d the pellet of cell debris. 100ul of 50% slurry Ni -agarose beads in lysis buffer was added to the crude cell supernatant and mixed on a shaker at roo m temperature for an hour and suspension was centri fuge at 1500rpm for 1 minute and supernatant was collec ted as unbound fraction and labelled as UF for SDS PAGE. The pelleted agarose beads can be wash by adding 1ml of wash buffer and let it rest for 5 minutes follow by cen trifuge f or 30 seconds at 1500rpm then transfer the bulk of su pernatan t to fresh microfu ge and label as wash for SDS page. Washing steps was repeated for 3 times , follow up by resuspend the washing beads in 250u l of elution buffer and incubate at room temp erature for 5 minutes allow the release of hexahistidine -
tagge d protei n. The rest of the pellet was resuspended in elution buffer for 5 minutes at room temperature and spin at 1500rpm for 1 minute , supernatant was collected as eluted fraction and prepare f or SDS PAGE analysis. Analysis using SDS -PAGE and IMAC Fracti on sampl es. IPTG induction time course sample was prepared by using 200ul /OD600 by resuspending pellets in water and loading buffer of 1 to 1 volume , follow up by IMAC sample preparation by addi ng loading buffer in 1 to 1 volume as well. 20ul of both sampl e is the n insert into 10% SDS-PAGE with Coomassie Blue staining . RESULTS (500 words max excluding figures/capt ions) Figure 1. is the result of PCR product obtained from the SYBR SAFE stain ed agaro se
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