Identification, Purification and Quantification of GST/GFP and GFP

Title: Successfully identify the mutations of GFPS65T and purification of GFP.

Abstract: GFP (Green fluorescent protein) is a useful reporter protein in the study of the interest gene expression. The mutant plasmid pGEX-GFPS65T-1 and pGEX-GFPS65T-8 are used in this experiment to study the impact of mutation in the ability of fluorescing. Unknown colony that contained pGEX-2T pGEX-GFPS65T-1 and pGEX-GFPS65T-8 will be identified and characterized by PCD and fluorescing. to fluorescing. The digestion and PCR of pGEX-GFPS65T-1 and pGEX-GFPS65T-8 show they had same plasmid size. In fluorescent scoring, pGEX-GFPS65T-1 showed a good capability in fluorescing while pGEX-GFPS65T-8 lose the capability. The mutation of S65T and T65A on amino acid position 65 are found which have a big impact and contribute for the fluorescent capability of the protein. The GFP that used for purification will be in a form of fusion protein with GST (Glutathione S-transferase) while using E.coli as a production cell. Purification of the fusion protein will be done by affinity column that contain immobilized glutathione. Purification of GFP from GST-GFP is done by thrombolytic cleavage. The purification yield and quality will examined by SDS-PAGE, Bradford Protein Assay, CDNM(1-chloro-2,4,-dinitrobenzene), and UV spectrometry. The purification and isolation of GFP from this method and give a yield of 8.417% based on the mass of fusion protein and GFP from CDNM and Bradford Protein Assay.

Introduction:

 Green fluorescent protein (GFP) is a great reporter protein that isolated from jellyfish Aequorea Victoria due to its ability to fluorescent[1]. The expression of GFP do not require any substrates or cofactor to its fluorescence and is able to fluoresce on both prokaryotic and eukaryotic including Escherichia coli, Caenorhabditis elegans, plant and mammalian cells[1-3]. Therefore, GFP is a useful reporter protein for indicating gene expression and localization of gene in organism/cells.

 There are 238 amino acids in GFP while amino acids 65,66, and 67 are important for the fluorescent ability. The natural form of GFP emits green fluorescent under UV light which peaked at 508nm[4]. However, the protein folding of wild type GFP (wtGFP) only efficient below room temperature and the folding efficiency falls steeply in higher temperature[4]. Therefore, this limiting factor was difficult to detect in mammalian cells due to its poor expression in emitting green light. In 1996, 2 mutant GFPs were found by Cheng which is GFP65T and RSGFP4 which are 18-fold and 24-fold brighter than wtGFP[2]. There is a single substitution mutation in GFP65T from serine to threonine and allow it to emit up to 18 times brighter green light in mammalian cells[2].

 In this experiment, Glutathione S-transferase(GST) from Schistosoma japonicum will be used as a fusion partner for GFP while pGEX will be used as a vector. The fusion protein is designed as GST in N-terminal while GFP located at C-terminal.  GST serve as a protein tag in this experiment and allow the purified of GFP protein by affinity chromatography with immobilized glutathione. The elution of GFP protein from affinity chromatography is done by thrombolytic cleavage after the washing of column.

 The mass of GST/GFP fusion protein will be determined by the enzymatic activity of GST in CDNM assay. Also, the mass of GFP after purification will be determined by both UV spectrometry and Bradford Protein Assay(BPA). The product yield of GFP from GST/GFP protein will then be calculated based on the difference on mass.

Experimental procedure:

Section 1: Identification, Confirmation and Screening of pGeX expression vector.

1.1: PCR, electrophoresis and fluorescent scoring of unknown pGEX plasmid.

Aim: To identify and characterize unknown plasmid by PCR and fluorescent scoring and compare with known recombinant pGEX plasmids. Also, the composition of the unknown plasmids in unknown plate will be determined based on the pooled result from the class.

Method: Refer to BIOC6017 lab manual part I page 15-18. The master mix recipe is shown in table 1.

Section 1.2: The endonuclease digest by restriction enzyme.

 Aim: To study and analyze the restriction enzyme site in recombinant pGEX plasmid and compare the result with known recombinant pGEX plasmids based on restriction enzyme digestion

Method: Refer to BIOC6017 lab manual part I page 19-20. Recipe for BamHl/ Pstl and EcoRl/Pst l are shown in table 2 and 3

Section 1.3: cDNA sequence analysis of recombinant pGEX plasmids

Aim: To analysis how the mutation in molecular level affect the ability of fluorescent among various GFP-encoding gene.

Method: Refer to BIOC6017 lab manual part I page 20.

Section 2: Purification, qualification and quantification of GST/GFP fusion protein and GFP.

Section 2.1: Purification of GFP.

Aim: To purify GST/GFP fusion protein from pGEX-GFP transformed E.coli TP1000 and further purify GFP from GST/GFP fusion protein. The obtained GST/GFP fusion protein and GFP will be collected and used examine the efficiency of purification.

Method: Refer to BIOC6017 lab manual part I page 23-26.

Section 2.2: SDS-PAGE analysis

Aim: To analyze the protein composition before, after and during purification qualitatively.

Method: Refer to BIOC6017 lab manual page 26-28.

Section 2.3: UV spectrometry for quantifying GFP.

Aim: To quantify obtained GFP via UV spectrometry and Beer-Lambert Law.

Method: Refer to BIOC6017 ab manual page 28.

Section 2.4: Bradford protein assay

Aim: To quantify the amount of purified GFP obtained and compare with the result in UV spectrometry.

Method: Refer to BIOC6017 lab manual 29-30. Preparation of Bovine Serum Albumin(BSA) standard is shown in table 4.

Section 2.5: CDNB assay

Aim: To determine to amount of GST/GFP fusion protein based on the enzymatic activity of GST.

Method: Refer to BIOC6017 lab manual page 31-32. Preparation of GST-GFP standard is shown in table 5.

Result

Section 1.1:

 The result of this section is showed in figure 1, table 6, and table 7. In figure 1, 3 bands with size~850bp are identified in all 3 unknown sample. The same size of band (~850bp) is also observed in pGEX-GFPS65T-1 and pGEX-GFPS65T-8. According to table 6, unknown plasmid 1 and 2 are able to fluoresce while unknown plasmid 3 is unable to fluoresce. Based on the result of table 7, the class compositions of different plasmids are determined as 81.61% of pGEX-GFPS65T-1, 9% of pGEX-GFPS65T-1, 0% of pGEX-2T and 9.5% of data not fitting into those 3 classes.

Section 1.2:

 The predicted fragments size from both digestions are listed in table 8. The result of gel electrophoresis and restriction enzyme are showed in figure 2. Lane 2 and 3 both give similar bands around 1kbp and 4kbp which matched with the predicted size of digestion for pGEX-2. Lane 5, lane 6, lane 8 and lane 9 which represented digestions for pGEX-GFPS65T-1and pGEX-GFPS65T-8 have same double bands which have ~1kbp and 4.7kbp for EcoRl/Pstl digest and ~1.7kbp and 4kbp for BamHl/Pstl digestion. This mean that is no big fragment insertion or deletion happen on pGEX-GFPS65T-8 which cause it to not fluoresce. Also, the sequence size and restriction enzyme site of pGEX-GFPS65T-1 and pGEX-GFPS65T-8 are the same according to figure 2. The result of all 3 non-digested group showed 3 bands which is out of expectation and will be discuss in discussion.

Section 1.3:

 No mutation is found among GFPS65T and pGEX-GFPS65T-1. There were 3 different site of amino acids found between WTGFP and synthetic GFPS65T which is insertion on WT position 1, substitution of Serine to Threonine on site 65 and substitution of Histidine to Leucine on site 231. According to the introduction of lab manual, the site that contribute to the mutation which led to a 6 times brighter on synthetic GFP is the mutation on site 65. There is one nucleotide mutation in position 196 of pGEX-GFPS65T-8 which contribute to the mutation of Threonine to Alanine when comparing the sequence of pGEX-GFPS65T-8 with synthetic GFP.

Section 2.1:

 The sample during purification and after purification will be sent for the qualitative and quantitative analysis of GST-GFP and GFP by using UV spectrometry, CDNM assay, and Bradford protein assay.

Section 2.2:

 In the result of SDS-PAGE, there is only one visible band in purified GFP lane and have a size of around 27kDa which is expected. This result is not expected while the expected result will be multiple fainted bands on uninduced sample, multiple fainted band with one intense band on induced sample which represent GST-GFP fusion protein, two intense bands on 50% slurry which represent GST-GFP fusion protein and GFP, and one intense band around 27KDa which represent purified GFP.

Section 2.3:

 The concentration and the yield of purification of GFP will be calculated by using Beer-Lambert law based on the result of UV spectrometry. Calculations are shown below while a final mass of GFP is 405 µg.

$A_{280}\mathit{obtained}:$

0.197A

$\mathrm{\epsilon }=19890$

Molecular weight of GFP: 26.9KDa

$A_{280}=\mathrm{\epsilon IC}$

0.197= 19890 x 1x C
C=9.904 uM

Concentration: 9.9uM x26900 g/mol =0.270µg/ul

Total mass: 0.27µg/ul x 1500ul= 405µg

Section 2.4: 

 The raw data is summarized and show in Table 9 while the concentration of the standards and the average value absorbance readings are used to plot into a BPA standard graph. The mass of GFP based on BPA will then calculate as shown below. The final mass of GFP is determined as 29.625 µg.

Equation obtained from standard Plot: Y =0.0085x +0.1387

$C_{1:10}=0.418$

$C_{1:10\mathit{ diluted}}$

= $\frac{0.273–0.139}{0.085}$

$C_{1:10\mathit{ diluted}}=1.580\mathit{ug}/\mathit{ml}$

Sample is 1:10 diluted, $C_{\mathit{final}}=15.800\mathit{ug}/\mathit{ml}$

Total mass of purified protein:

1.5ml x 15.8µg/ml x $\frac{500}{400}(400\mathit{ul\; of\; diluted\; sample\; is\; added\; to} 100\mathit{ul\; BSA\; reagent}$

)

= 29.625µg

Section 2.5

 The raw data from standard obtained is presented in table 10. The standard group of 4µg GST-GFP is an outlier and not been used in the further analysis. The absorbance of standard will be plot against time and each standard slope will be obtained and plot against each concentration of standard and shown in figure 5. The raw data of samples in CDNM assay is shown in table 11 while the average value of the samples is shown in table 12. The GST-GFP amount in uninduced sample show same expression as in 0µg GST-GFP standard which mean the expression of GST-GFP in uninduced sample is very close to zero. The slope of sample will also be determined and plot into standard slope vs concentration graph to obtain the concentration of sample and shown in figure 6. The calculation for the mass of GST-GFP is shown below. A GST-GFP mass of 351.91µg was determined from 1:10 diluted sample with a total amount of 0.5ml of induced cell lysate.

Slope of sample: Induced= 0.0258
    Uninduced= 0.008

Equation from standard plot: Y=21.8x X0.012

0.0258= 21.8 x $C_{\mathit{induce\; in\; cuvette}}$

+0.012

$C_{\mathit{induce\; in\; cuvette}}$

= 6.33 x ${10}^{–4}$

µg/ul

$C_{\mathit{Original}}=6.33\mathrm{ x }{10}^{–4}\mathrm{\mu g}/\mathrm{ul }$

x100 (10uL of sample in 1000uL reaction)

                  = 6.33 x ${10}^{–2}$

µg/ul

Mass of GST-GFP: 6.33 x ${10}^{–4}$

µg/ul x 5560ul

Mass of GST-GFP: 351.950µg

Discussion:

Section 1.1

All 3 unknown samples are identified as pGEX-GFPS65T-1 or pGEX-GFPS65T-8 according to the examination of PCR result as they show a same band size as the pGEX-GFPS65T-1 or pGEX-GFPS65T-8 plasmid. According to fluorescent scoring, only pGEX-GFPS65T-1 able to fluoresce when compared with two other plasmids. Hence, unknown 1 and unknown 2 are successfully identified as pGEX-GFPS65T-1 as its PCR band size indicate pGEX-GFP plasmid while it is able to fluoresce. Unknown 3 is identified as pGEX-GFPS65T-8 as its PCR band size indicate pGEX-GFP plasmid and its unable to fluoresce. The composition of the unknown plate is 81.61% of pGEX-GFPS65T-1, 9% of pGEX-GFPS65T-1, 0% of pGEX-2T and 9.5% of data not fitting into those 3 classes.

Section 1.2:

The result of all 3 non-digest group showed multiple bands in all groups of Amir and is out of expectation. A contamination of the non-digest sample/ during the digesting procedure could be the reasons of this result. A repeated, further experiment or study would be necessary to address this problem.

Section 1.3:

All observed mutation is matching with the literature while the Serine in position 65 is further proved to serve as important function in the ability to fluoresce. Also, mutation of position 65 into Alanine in GFP contribute to the loss of fluorescing ability in pGEX-GFPS65T-8.

Section 2.2:
The result of SDS-PAGE is not matching with expected result as discussed in result section. The main reason of this is mostly because the concentration of the sample is too low. Also, the staining colour of the gel of my group is much lighter than other groups which might be one of the reason that causing the fainted bands.

Section 2.3, 2.4 & 2.5:
A final mass of GFP is 405 µg from UV spectrometry, 29.625 µg from BPA while the final mass of GST-GFP is determined as 351.95µg. The result of UV spectrometry is bias high in this experiment as it is even higher than the mass of GST-GFP. This might because there is nucleic acid in the sample as nucleic acid also absorb in 280nm or the interference of chromogenic substrates. Hence, the result of the BPA is more reliable in this case as the dye can successfully bind to GFP protein. A calculation of the product yield of GFP from GST-GFP based on mass from BPA is shown below. A yield of 8.417% is obtained from the purification.

Yield= $\frac{29.625\mathit{ug}}{351.95\mathit{ug}}$

x100%
         = 8.417%

References:

1. Chalfie, M., et al., Green fluorescent protein as a marker for gene expression. Science, 1994. 263(5148): p. 802-5.

2. Cheng, L., et al., Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat Biotechnol, 1996. 14(5): p. 606-9.

3. Chiu, W., et al., Engineered GFP as a vital reporter in plants. Curr Biol, 1996. 6(3): p. 325-30.

4. Tsien, R.Y., The green fluorescent protein. Annu Rev Biochem, 1998. 67: p. 509-44.

Table

Table 1:Master Mix recipe for PCR.

Table 2:Components for BamHl/Pstl digestion.

Table 3: Components for EcoRl/Pstl digestion.

Table 4: Recipe for preparation of BSA standard.

Table 5: GST/GFP standard for CDNM assay.

Table 6:Collated result for fluorescence analysis in individual group.

Table 7: Collated class result based on PCR and fluorescence scoring.

Table 8:Calculations and predicted size of fragments for restriction enzyme digestion.

Table 9:Summarized table for BPA raw data.

Table 10: Raw data for CDNM standards reading.

Table 11: Raw data of Induced and Uninduced sample in CDNM.

Table 12: Average value of Induced and Uninduced sample in CDNM.

Figure legends:

Figure 1: Gel electrophoresis of PCR that contained 3 unknown samples, pGEX-2T, pGEX-GFPS65T-1, and pGEX-GFPS65T-8.

Figure 2: Result of restriction enzyme digestion.

Figure 3: SDS-PAGE of uninduced, induced,50% slurry, and purified GFP.

Figure 4: Plot of BPA standard result, the standard 5µg/ul is excluded as it is an outlier.

Figure 5: Absorbance vs time graph for CDNB standard.

Figure 6: CDNM standard slope vs standard concentration.

Figure 7: Absorbance vs time graph of induced and uninduced sample in CDNM.

Figure

Figure 2

Figure 3

Figure 4

Figure 5.

Figure 6

Figure 7