This report is presented as part of the completion of a credited internship at the Department of Biotechnology, Faculty of Applied Sciences, UCSI University in Kuala Lumpur. All details and findings of the research conducted during the internship period are as reported. The aim of the project was to express the recombinant Tiger Milk Mushroom Fungal Immunomodulatory Protein (FIP-Lrh) protein by using E.
Si, Ph.D for their support, guidance, supervision and valuable knowledge for the completeness of the project and report. Tiger Milk Mushroom's FIP-Lrh is a new FIP member believed to have pharmaceutical values such as anti-inflammatory, anti-allergy, anti-cancer, etc. However, functional studies have been hampered due to high cost and time required for direct purification of the mushroom protein.
The induction of expression culture with 1 mM IPTG resulted in an overexpressed 14.9 kDa soluble recombinant FIP-Lrh which was subsequently purified using the Ni-NTA purification system. The recombinant FIP-Lrh was successfully purified, with some weak non-specific binding of non-target protein to the NiNTA resin necessitating secondary purification. Further optimization for recombinant protein production needs to be done to obtain a sufficient amount of recombinant FIP-Lrh for downstream analysis.
Keyword: Fungal immunomodulatory proteins, tiger milk mushroom, recombinant protein, Escherichia coli expression system, Ni-NTA purification.
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Buffer, Reagents, and Solutions Preparation Luria Broth Preparation
Tris-HCl at a final concentration of 1.0 M was prepared by mixing 12.11 g of Tris powder with 70 mL of dH2O and the pH was adjusted to 6.8. Both Tris-HCl buffers were then made up to 100 mL with dH2O and autoclaved at 121oC for 15 minutes. SDS buffer with 10% concentration was prepared by dissolving 5g SDS powder with 30mL dH2O and then made up to 50mL.
10% ammonium persulfate (APS) was prepared by dissolving 1 g APS powder with 10 mL dH2O and aliquoted into several 1.5 mL microcentrifuge tubes before storage at −20 °C. Working liquid buffer (1X) was directly prepared by dissolving 200 ml of 5X liquid buffer with 800 ml of dH2O. 6X SDS dissociation buffer was prepared by mixing and vortexing 3.75 mL of 1M Tris-HCl, 5 mL of glycerol, 0.9 mL of β-mercaptoethanol, 1.2 g of SDS powder, and 0.012 g of bromophenol blue powder.
Working stock of 5X native purification buffer was prepared by dissolving 7g NaH2PO4 and 29.2g NaCl with 180mL dHO and was adjusted to pH 8.0 while washing the volume afterwards. While 1X native purification buffer or native binding buffer was prepared by diluting 40mL 5X native purification buffer with 140mL dH2O and adjusted to pH 8.0 and final volume made up to 200mL with dH2O. Phenyl methanesulfonyl fluoride (PMSF) stock solution (100mM) was prepared by dissolving 87.1mg PMSF with 5mL isopropyl alcohol and stored at -20oC.
Sonication buffer with 0.2 mM PMSF was prepared by diluting 60 µL of PMSF (100 mM) with 25 mL of 1X wash buffer and adjusting to pH 8.0 before making up to 30 mL with dH2O. A native elution buffer containing 250 mM imidazole and 10% glycerol was prepared by mixing 5 mL of glycerol and 4.17 mL of 3 M imidazole with 40 mL of 1X native purification buffer, adjusted to pH 8.0, and then made up to 50 mL with 1X natural purification buffer. The final volume was made up to 1 L with dH2O and autoclaved at 121 °C for 15 min.
BSA stock was prepared by dissolving 5 mg BSA powder in 5 ml dH20 to reach a final concentration of 1 mg/ml. The 15% dissolving gel was prepared as shown in Table 1, and poured directly into the casting frame leaving 1 cm above for gel separation. After solidifying the gel, 5% stacking gel was prepared as shown in Table 1 and then transferred to the casting frame and combed.
Purification of FIP-Lrh from cell lysate of flask 1b expression culture was performed using Ni-NTA affinity chromatography under native condition as described in section 2.1.4.3. SDS-PAGE analysis of the different fraction from different stages of purification is as shown in Figure 5. The FIP-Lrh protein band in the total lysate from sonication (Lane 1) was bound on the Ni-NTA resin if the FIP-Lrh band was absent in the flow-through fraction (Lane 2).
The non-target protein was eluted in 20 mM imidazole indicated by non-target band (lane 3). The 30 mM imidazole concentration showed neither non-target nor target bands (lane 4); indicating that recombinant FIP-Lrh was still bound to Ni-NTA. At 40 mM imidazole, a faint band of FIP-Lrh was present, indicating that some of the recombinant protein was eluted during the wash (lane 5).
The FIP-Lrh protein band appeared more intense in lane 6 to lane 10 eluted fractions as shown in figure 4. Some smear and non-target bands were observed on lanes 6 to 8. Also mentioned, purification was performed for pellet from the other flasks of expression culture. Dialysis was performed separately for eluent fractions containing non-target bands; the rest of the eluent fractions were combined and dialyzed together. -PAGE analysis of dialyzed recombinant protein. a) First SDS-PAGE analysis and (b) second SDS-PAGE analysis.
All dialyzed protein samples were pooled and concentrated into a fraction of 500 µL of concentrated sample. Concentrated protein samples at 10-fold dilution showed a prominent band of the purified recombinant protein with very weak non-target band (lane 2) (see Figure 6). Recombinant FIP-Lrh was present in the flow-through with an identical protein profile (lane 1) (see Figure 6).
The standard curve showed a high linear regression coefficient of determination (R2) of 0.9823, allowing the results of dependent variables to be used to determine the independent variable, i.e. the standard curve was reliable.
FIP-Lrh Protein Concentration
Protein Sonication and Purification
The PMSF, a protease inhibitor, was added to the lysis buffer to prevent the degradation of the recombinant protein by serine proteases such as chymotrypsin and trypsin (Nichols et al., 2020). Protein degradation can be analyzed via SDS-PAGE, which is indicated by the presence of a second protein band next to the target protein (Liao & Chen, 2015). In this study, the Ni-NTA purification system was used as the recombinant FIP-Lrh with 6xHis tag at the C-terminal (Ejike et al., 2021).
In addition, the recombinant FIP-Lrh bound to the nickel could be eluted by adjusting the pH of the buffer or adding free imidazole that would compete for the binding with nickel. The disadvantage of using a polyhistidine tag is the likelihood of nonspecific binding of untagged protein due to the possibility of cellular proteins containing two or more adjacent histidine residues (Bornhorst & Falke, 2000). In this study, 250 mM imidazole was added to the column and eluted fractions of recombinant FIP-Lrh were analyzed using SDS-PAGE.
The protein flow-throughs from different batches showed little or no target recombinant protein bands (see Figure 4 & Appendix 2). Optimizing the amount of imidazole concentrations used (20 mM, 30 mM and 40 mM) in wash buffer could help to increase the stringency of the washes and thus remove unwanted proteins bound to the Ni-NTA resin without washing out the target protein (see Figure 4). Protein dialysis was used to remove salts and imidazole from purified recombinant FIP-Lrh (Harcum, 2008).
According to the results shown in Figure 5, there was no protein smearing for eluted fractions after dialysis, since smearing could also occur due to the combination of high salt concentration and protein overload. It was later found that 90.83% of FIP-Lrh protein was found in the flow-through (see Figure 6) during the concentration process due to the use of high centrifugation speed (10,000 rpm) applied to the centricon used. Fortunately, the flow-through was not discarded and could be used for re-concentration of FIP-Lrh using a new centricon and at lower centrifugation speed.
According to (Ejike et al., 2020), the recombinant protein product can potentially be contaminated with endotoxin and the absence of post-translational modification; render protein non-functional. However, since the recombinant protein would only be used for protein crystallography, such a problem is neglected. Furthermore, the presence of recombinant FIP-Lrh in the flow-through during concentrating process resulted in less purified FIP-Lrh (6.54mg) obtained for secondary purification using size exclusion chromatography before protein crystallization.
CONCLUSION AND RECOMMENDATION
SELF REFLECTION
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