Applied Molecular Biology

Isolation and Characterization of Plasmids and Recombinant Proteins

Isolation and Characterization of Plasmids and Recombinant Proteins (Lab Report)


Protein purification is the first step towards the determination of protein function. To determine protein function, protein is heterologously expressed in E. coli before purification and characterization. Interestingly, a fusion tag such as GST is attached to isolate proteins of interest from the other proteins present in E. coli to facilitate affinity-based purification. Whereas GST enhances purification, other tags, such as GFP, are fluorescent and facilitate protein localization/compartmentation determination. The present study extracted a plasmid containing two fusion proteins, pGFP-GST, from the E. coli HB101 strain. The plasmid was subsequently characterized by PCR using GST-specific primers, resolved using 1% agarose gel and stained with ethidium bromide. Plasmid concentration was determined spectrophotometrically, while DNA purity was accessed by measuring Abs260/280. Following heterologous expression in E. coli, recombinant protein was purified by means of the GST tag, while the GFP tag facilitated the determination of protein localization. After affinity purification, protein purity was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie staining. Data from agarose gel electrophoresis shows the expected DNA size for GST (1415 bp), suggesting that the plasmid contains a GST DNA sequence. The plasmid concentration was determined to be 12.79µg/mL and was determined to be pure (Abs230/280 =1.884/2.671). Data from the GST-affinity purification shows the purified protein has an estimated size of 42kDa, which is lower than the expected size (55.3kDa). This may have been due to proteolysis during the lysis step. In conclusion, the plasmid encoding the pGFP-GST fusion protein was successfully purified and characterized.


Proteins play important roles in regulating body metabolism in normal and disease conditions. To specifically determine the function of a protein, the protein’s DNA sequence is cloned into a vector to form a plasmid, which is then transformed into Escherichia coli to produce the recombinant protein. After overexpression in E. coli, the recombinant protein is subsequently purified, and the pure protein is characterized using its inherent features such as activity, shape, size, and charge (Tan et al., 2009).

Protein purification is critical to separating the recombinant protein of interest from the multitude of unwanted proteins present in the bacterial strain. To facilitate protein purification via affinity chromatography, affinity tags such as hexahistidine tag (6xHis), maltose binding protein (MBP), and Glutathione-s-transferase (GST) are usually attached to the C-terminal or the N-terminal end of a protein of interest (Kimple et al., 2013). Besides purification, affinity tags such as MBP, thioredoxin (trxA), and GST tags also enable the expression of recombinant proteins (Kimple et al., 2013). Multiple studies have established the role of GST tags in the expression and purification of recombinant proteins (Young et al., 2012; Schäfer et al., 2015; Kachel, 2016). GST plays an important role in the phase II detoxification pathway, where it conjugates glutathione to xenobiotics, enhancing their solubility and clearance (Allocati et al., 2018). Previous studies have established that GST enhances the solubility of its passenger protein by acting as a molecular chaperone, thereby avoiding the entrapment of recombinant proteins in inclusion bodies (Chen et al., 2015).

Beyond enhancing protein expression and purification, tags can also aid in studying protein kinetics, localization, and investigation of protein-protein interaction. An example of such a tag is the fluorescent tag – green fluorescent protein (GFP). As a fusion protein, GFP is a reporter gene or selectable marker useful for monitoring real-time protein interactions (Rizzo et al., 2010; Kremers et al., 2010). The GFP is a homologous protein derived from chromophore-carrying marine organisms such as the jellyfish Aequorea victoria. The chromophores are located in a few of the amino acids within A. victoria. Due to its genetically encoded chromogenic and autocatalytic abilities, GFP fluoresces effortlessly in both the prokaryotic and eukaryotic cells (Pakhomov et al., 2008) and can therefore be used to study protein localization. Given the individual merits of GST-tag and GFP-tag, the presence of both tags in a fusion protein could potentially enable protein expression/purification and characterization of protein localization and kinetics.

The present study extracted a plasmid pGFP-GST from the E. coli HB101 strain. The plasmid was subsequently characterized by PCR using GST-specific primers. Following heterologous expression in E. coli, the recombinant protein was purified by means of the GST tag, while the GFP tag facilitated the determination of protein localization.

This study has two aims: A) To determine the localization of Glutathione-S-Transferase (GST) in the protein of interest using the GFP tag. B) To characterize the recombinant protein extracted via affinity purification using the GST tag.


Isolation and Characterization of Plasmid pGFP-GST

Coli HB101 strain utilized in this experiment was obtained from Prof. Richardson’s laboratory. The plasmid DNA-carrying gene constructs encoding GFP, and GST genes were extracted from an E. coli bacterial culture. The process involved centrifugation of 1mL bacteria at 12000rpm for 1 minute. After this, the resultant supernatant was discarded, and the step was repeated thrice to ensure the successful extraction of cell pellets. Protocols from the QIAprep Miniprep Handbook were utilized for further purification of plasmid DNA (Qiagen Corporation, 2005). The plasmid DNA concentration and ratio (OD260/280 and OD230/280) were measured by testing 2µL of plasmid DNA on a Biodrop. Furthermore, diluted template DNA was amplified via PCR using primers (GFP-CT GST-NT and GST-CT GFP-NT) and 12.5µL PCR buffer containing the DreamTaq DNA polymerase (from DreamTaq Master Mix by Thermofisher). Sterile water was added to make up the desired volume to maintain the expected final concentration of reactants. PCR products were resolved on a 1% agarose gel. The gel was subsequently stained with ethidium bromide and visualized with UV light.

Isolation and Characterization of Recombinant GFP-GST Protein

Bacterial lysate obtained from the overexpressed recombinant plasmid was affinity-purified to isolate further and characterize the recombinant GFP-GST protein. To achieve this, a Glutathione Sepharose Column was prepared by placing 500-750µL bed volume Glutathione Sepharose 4B gel into a poly-prep column. The column was equilibrated with 30 mL of Polyphosphate Buffered Saline (PBS). The lysate was loaded into the column, and the flowthrough was collected in a sterile 50 mL Falcon tube. This was repeated twice to allow the percolation of bacterial lysate through the column. Next, the column was washed with 30 mL PBS to remove unbound lysates. Finally, the recombinant protein was eluted from the column with 500 mL of reduced glutathione solution. 500 µL fractions were collected, and protein concentration (A260/A280) was measured on the Biodrop.

To measure the molecular weight of the eluted proteins, 5µg protein was added into 10µL Laemmli running buffer containing 10% of the reducing agent, ß-mercaptoethanol (BME). The solution was resolved on a 4-20% Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS-PAGE). After electrophoresis, the gel was stained with Coomassie dye, de-stained and then scanned.


Isolation and Characterization of Plasmid pGFP-GST

Plasmid DNA-carrying gene constructs GFP-GST was extracted from E. coli cells through affinity chromatography. The spectrophotometric concentration of the isolated plasmid DNA was 12.79µg/mL. DNA purity was assessed by measuring Abs260/280 and Abs230/280, which was found to be 1.884/2.671. A standard PCR reaction using two primers was performed to determine the expression of heterologous GFP-GST genes from plasmid DNA. Upon gene expression, affinity chromatography using DNA agarose gel electrophoresis was performed to determine the size and positions of the two PCR products on the amino acid sequence (Fig. 2) and the plasmid DNA (Fig. 3). The analysis was conducted with samples from seven researchers each loaded into a separate gel lane in order to be able to compare results. The gene constructs served as the positive PCR controls.

Based on gel electrophoresis results, PCR amplification of plasmid DNA was successful. As can be seen on the gel, the size of the band of the PCR product is comparable to the expected size (1415 bp). However, the correct size of the PCR products could not be estimated because the ladder used (1kb) was not well resolved. The results could have been better if a 50bp ladder was used. The accurate yield of DNA may be estimated using the formula:

(M2/M1) x 100%, where M2 = final yield; M1 = initial yield

Additionally, the position of the two red-dotted lanes on the PCR screen in fig. 2 above indicates that GFP is in the upstream position.

Furthermore, the size of the band of the plasmid is also comparable to the expected size (2436bp). In this case, the ladder was well resolved and gave accurate readings of the gel.

Isolation and Characterization of Recombinant GFP-GST Protein

Analysis of bacterial lysate derived from the isolated plasmid was conducted via SDS-PAGE to determine the size of the translated protein. The lysate was generated by transforming the plasmid into E. coli MC1061 strain and growing it in a selective media with ampicillin resistance and the reagent isopropyl-ß-D-thiogalactopyranoside (IPTG) to induce protein translation. Under French Press and PBS, the bacterial strain was lysed and centrifuged to achieve a clear lysate. Given that GFP and GST have molecular weights of 26.9kDa and 28.4kDa, respectively, the predicted molecular weight of the fusion protein, GFP-GST, is expected to be 55.3kDa.

Based on the SDS-PAGE results (fig. 4), the protein purification on lane 3 is purer than others, including my sample in lane 10. The assayed protein was observed to have a lower molecular weight of approximately 42kDa than the predicted molecular weight of the fusion protein. The proteins loaded into different gel lanes expressed differing electrophoretic mobility. Protein concentration was measured as 2323.6µg/mL, and protein purity was assessed by measuring Abs260/280, which was found to be -4.909.


Affinity tags such as GST enhance protein purification, while fluorescent tags facilitate protein characterization. In this study, GST and GFP were utilized to purify and characterise the protein of interest, respectively. PCR amplification and a subsequent agarose gel electrophoresis using primer cocktails (GFP-CT GST-NT and GST-CT GFP-NT) show the expected DNA size (Fig. 2). This suggests that the plasmid vector contains GST since the primers specifically amplified the PCR band corresponding to the DNA size of GST (1415 bp). However, the correct size of the PCR products could not be estimated because the ladder used (1kb) was not well resolved. The results could have been better if a 50bp ladder was used. The position of the GFP upstream on the PCR screens indicates that GST is positioned at the C-terminal end of the open reading frame (ORF) in the plasmid vector.

As seen on the SDS-PAGE gel (Fig. 4), GST-affinity purification of the recombinant protein was successful. This shows that the recombinant protein expresses GST in addition to its passenger protein. However, the purification by the researcher Drupal (lane 3) appears purer compared to other lanes. This is probably because Drupal washed more vigorously before the elution step. The protease inhibitor cocktail was used to prevent the degradation of the protein of interest in the event of proteolysis during protein purification. However, the apparent molecular weight of the protein recorded is 42kDa compared to the predicted molecular weight (55.3kDa). The difference in molecular weights of the proteins may be due to proteolysis or premature protein termination at the ribosome. Other factors contributing to this size difference may be extensive protein glycosylation and phosphorylation, formation of protein complexes, formation of aggregates, etc. (Sigma-Aldrich, 2018). Additionally, differential electrophoretic mobility was observed on the proteins on the acrylamide gel. This may be due to incomplete denaturation of the proteins by SDS. This may cause incomplete linearization and a weak negative charge in some proteins, resulting in slower migration through the gel (Rath et al., 2009). The protein concentration was recorded to be 2323.6µg/mL. Spectrophotometric protein estimation methods such as bio drop give reasonably accurate results due to the high sensitivity of the spectrophotometer. However, the protein concentration can be further validated by conducting a bicinchoninic acid (BCA) assay using Bovine Serum Albumin (BSA) standards of known concentration and comparing both protein concentrations. 


Characteristics of proteins such as charge, size, shape and activity are critical for successful protein expression and purification. Protein tags such as affinity (GST) and fluorescent (GFP) play important roles during these events. For instance, GST, localized at the C-terminal end of the protein of interest in this study, improved protein expression and purification. GFP also enhanced the localization and interaction of the GST protein during protein purification.  Isolation and characterization of this study’s plasmid and recombinant protein were successful as they mostly gave desired results.


Allocati, N., Masulli, M., Di Ilio, C. and Federici, L. (2018). Glutathione transferases: substrates, inhibitors and pro-drugs in cancer and neurodegenerative diseases. Oncogenesis, [online] 7(1). Available at:

Chen, X., Shi, J., Chen, R., Wen, Y., Shi, Y., Zhu, Z., Guo, S. and Li, L. (2015). Molecular chaperones (TrxA, SUMO, Intein, and GST) mediating expression, purification, and antimicrobial activity assays of plectasin in Escherichia coli. Biotechnology and Applied Biochemistry, [online] 62(5), pp.606–614. Available at: [Accessed 22 Apr. 2021].

Cloning vector pGEX3x GST-GFP gene, complete cds. (2012). NCBI Nucleotide. [online] Available at: [Accessed 20 Apr. 2021].

Kachel, W. (2016). Applications of the GST-Affinity Tag in the Purification and Characterization of Proteins. [online] . Available at:

‌Kimple, M.E., Brill, A.L. and Pasker, R.L. (2013). Overview of Affinity Tags for Protein Purification. Current Protocols in Protein Science, [online] pp.9.9.1–9.9.23. Available at:

Kremers, G.-J. ., Gilbert, S.G., Cranfill, P.J., Davidson, M.W. and Piston, D.W. (2010). Fluorescent proteins at a glance. Journal of Cell Science, [online] 124(2), pp.157–160. Available at: [Accessed 3 Nov. 2019].

Pakhomov, A.A. and Martynov, V.I. (2008). GFP Family: Structural Insights into Spectral Tuning. Chemistry & Biology, 15(8), pp.755–764.

Perkins, E.J. (2014). Plasmids 101: Protein tags. [online] Available at:

‌Qiagen Corporation (2005) QIAprep® Miniprep Handbook For purification of molecular biology grade DNA (2nd Ed)

Rath, A., Glibowicka, M., Nadeau, V.G., Chen, G. and Deber, C.M. (2009). Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences, [online] 106(6), pp.1760–1765. Available at:

Rizzo, M.A., Davidson, M.W. and Piston, D.W. (2009). Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells. Cold Spring Harbor Protocols, 2009(12), pp.pdb.top64–pdb.top64.

Schäfer, F., Seip, N., Maertens, B., Block, H. and Kubicek, J. (2015). Purification of GST-Tagged Proteins. Laboratory Methods in Enzymology: Protein Part D, [online] pp.127–139. Available at:

Sigma-Aldrich. (2018). Western Blotting: Why Are Observed and Calculated Molecular Weights Different? [online] Available at:

Tan, S.C. and Yiap, B.C. (2009). DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology, [online] 2009, pp.1–10. Available at: (2021). DreamTaq Green PCR Master Mix (2X). [online] Available at:

Young, C.L., Britton, Z.T. and Robinson, A.S. (2012). Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnology Journal, 7(5), pp.620–634.

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