Materials and Methods
Section 4 Results and Discussion
4.2 Bacterial Expression, Purification and Physical Characterization of the Recombinant Proteins
Upon confirmation of cloning, the expression of the evaluated proteins was induced. The expression was first optimized in small scale 5 ml cultures. PGEX‐4T‐2 vector system contains the Lac Z gene, which makes it an Isopropyl-β-D-1-thiogalactopyranoside (IPTG) inducible vector system. The transformed bacteria were grown on LB agar plates containing ampicillin. Primary culture of 3 ml grown from isolated single colony overnight at 37 °C was used as an inoculum for secondary culture of 5 ml LB media. The protein expression was induced by IPTG at different temperatures for different time periods. The expression conditions optimized for GST‐PTEN‐Long protein were 0.5 mM IPTG concentration, 12 h induction time at a temperature of 21 °C. Optimal conditions for expression of the
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68 Figure 4.1 Cloning of PTEN and PTEN‐Long genes (A) Lane 1 shows 1 kb DNA ladder, Lane 2‐4 shows digestion of pGEMT‐PTEN by EcoRI (B) Lane 1 shows 1 kb DNA ladder, Lane 2 shows the uncut PGEX-4T-2 plasmid and Lane 3 shows restriction digestion of PGEX-4T-2‐PTEN by BamHI and XhoI (C) Lane 1 shows 1 kb DNA ladder, Lane 2‐3 shows uncut digestion pGEMT‐PTEN‐Long, Lane 4 shows restriction digestion of pGEMT‐PTEN‐Long by EcoRI (D) Lane 1 shows 1 kb DNA ladder, Lane 2‐4 shows restriction digestion of PGEX-4T-2‐PTEN‐Long by BamHI and XhoI.
recombinant GST‐PTEN were 0.5 mM IPTG concentration, 4 h induction time at a temperature of 28 °C. The expression of the induced recombinant proteins were analyzed on 12% SDS gel (Figure 4.2A and 4.2B).
For both the recombinant proteins, at small scale of 5 ml secondary cultures when the cells were lysed and analyzed on SDS gel, most of the induced protein was found to be in the insoluble fraction with a small amount in the soluble fraction. However, upon increasing the culture volume to 500 ml, considerable amount of induced recombinant protein was found in the soluble fraction. Therefore, the supernatant fraction was used without solubilizing the
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69 Figure 4.2 Induction of PTEN and PTEN‐Long proteins expression (A) Lane 1 shows 2‐212 kDa protein ladder, Lane 2,4,6 displays supernatant fraction of induced cell lysate of different clones, Lane 3,5,7 displays expression of GST‐PTEN in pellet fraction of induced cell lysate of different clones (around 81 kDa) and Land 8 shows uninduced cell lysate (B) Lane 1 shows 2‐212 kDa protein ladder, Lane 2,6,8 displays supernatant fraction of induced cell lysate of different clones, Lane 3,7,9 displays expression of GST‐PTEN in pellet fraction of induced cell lysate of different clones (around 81 kDa) and Land 4,5 shows uninduced cell lysate.
pellet so as to avoid the use of detergents during the course of purification. The bacterial cells from 500 ml culture were harvested by centrifugation at 7000 rpm for 8 min and stored at ‐20
°C until further use. The frozen bacterial pellet were lysed and processed for purification.
Purification was carried out using glutathione‐agarose affinity chromatography. The protein was purified on ice under slow rocking conditions by following binding, washing and elution steps. A band of around 100 kDa and 81 kDa corresponding to GST-PTEN‐Long and GST- PTEN, respectively was observed when the purified fraction was run on 12% SDS-PAGE (Figure 4.3A and 4.3B). Protein yield determined by Bradford assay was approximately 5 to 10 mg per litre of E.coli culture for the recombinant proteins. GST tag was also excised by on‐column thrombin cleavage and the cleaved fractions were analyzed on 12% SDS-PAGE (Figure 4.3C and 4.3D). The purified recombinant proteins were further confirmed by MALDI TOF-TOF and Western blot analysis. The MS/MS profile obtained by MALDI TOF‐TOF analysis of trypsin digested peptide fragments inspected using findpept expasy tool, generated match of the peptide sequence EKVENGSL with sequence query coverage of 14% for GST‐PTEN‐Long protein.
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70 Figure 4.3 Purification profile of PTEN and PTEN‐Long (A) Lane 1 shows 2-212 kDa protein ladder and Lane 2 shows purified GST-PTEN (B) Lane 1 shows 2-212 kDa protein ladder and Lane 2 shows purified PTEN (C) Lane 1 shows 2-212 kDa protein ladder, Lane 2 and 3 flow through fractions, Lane 4 purified GST‐PTEN‐Long (D) Lane 1 shows 2-212 kDa protein ladder and Lane 2 shows purified PTEN‐Long.
For GST‐PTEN protein the peptide sequence match NDLDKANKDKANRYFSPNFKVK was generated with sequence query coverage of 16.13% (Figure 4.4A and 4.4B). Protein purified to homogeneity was investigated by the Western blotting using anti‐PTEN antibody and anti‐GST antibody. HRP‐conjugated with the secondary antibody was used to develop the blots, which generated bands at legitimate molecular weight of 100 kDa and 81 kDa corresponding to GST-PTEN‐Long and GST-PTEN, respectively. For GST band was developed at legitimate molecular weight around 26 kDa (Figure 4.4C and 4.4D).
Following bacterial purification, it is important to study the secondary structure integrity of the recombinant proteins. Secondary structure of GST-PTEN‐Long and GST-PTEN was characterized by far‐UV circular dichroism spectroscopy. The spectrum investigated by Yang’s reference (Chen and Yang 1971), revealed the content of secondary structural elements to be 45.5% α‐helix and 25.5% β‐sheet for GST-PTEN‐Long. The spectrum investigated revealed the content of secondary structural elements to be 24.5% α‐helix, 25.6
% β‐sheet for GST-PTEN, as determined by Yang’s reference (Figure 4.5A and 4.5B).
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71 Figure 4.4 Characterization of the recombinant proteins (A) MALDI TOF‐TOF analysis of purified GST-PTEN (B) MALDI TOF‐TOF analysis of purified GST-PTEN‐Long (C) Western blot with Anti-PTEN antibody and Anti-GST antibody for GST‐PTEN (D) Western blot with Anti-PTEN antibody and Anti-GST antibody for GST-PTEN‐Long.
The result was in accordance with the available crystal structure of PTEN in PDB (1D5R), where the estimated secondary structural elements were 19.603 % α‐helix and 24.5 % β‐sheet. Once the secondary structure was intact as evident by circular dichroism, the function of the recombinant enzymes were determined by phosphatase assay.