Research Article - (2020) Volume 0, Issue 0
Received: 28-Sep-2020
Published:
31-Oct-2020
, DOI: 10.37421/1747-0862.2020.14.S5
Citation: Chung, Choi Man, Ma CHY, Lai ATL, and Lin J, et al. “Expression of Human Epidermal Growth Factor in Escherichia coli by Intein Approach.” J Mol Genet Med 14 (2020): S5 doi: 10.37421/jmgm.2020.14.S5
Copyright: © 2020 Choi MC, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Different approaches are used to express recombinant proteins without the requirement of post-translational modification in Escherichia coli (E. coli). Though E. coli may have a drawback in producing endotoxin, its short division time and high expression of the final product are significant in making use as a host to produce various recombinant proteins. Inteins have been discovered in multiples microorganisms in facilitating the expression of homologous proteins and it has been used as one of the crucial tools for the production of recombinant proteins due to its unique feature in auto-excising its fusion partner, which also known as exteins. In this communication, we employed a well-established gp41-1 mini-intein to facilitate the expression of epidermal growth factor (EGF). The study revealed that though the epidermal growth factor cannot be excised from the gp41-1 mini intein during the expression, it showed the capability of gp41-1 mini intein in processing intracellular expression of soluble EGF fusion protein. Different conditions for inducing the cleavage of exteins from inteins has been studied by many research groups, and reducing condition by using the DTT works well on the C-terminal cleavage of EGF from the gp41-1 mini intein. The final purified, different concentration of EGF was mixed with homemade aqueous cream and showed to be highly active in accelerating the healing rate of patients suffering from bedsores, diabetic foot ulcers and skin rupture.
Epidermal Growth Factor (EGF) • Inteins • Healing
Epidermal growth factor (EGF) is a 53 amino acid oligopeptide with three disulfide bonds, which has been discovered 60 years ago. EGF can bind on the EGF receptor; thereby activate the downstream signalling transduction cascade in triggering the proliferation of epidermal cells. It has been shown that EGF works well not only in the improvement of wound healing, but also participates in various physiological pathways like tissue and bone regeneration [1-4].
Epidermal growth factor has a high affinity towards epidermal growth factor receptor (EGFR). The EGFR is located at the cell surface, which are inactive in monomeric form. Upon binding to EGF, the monomeric form undergoes transition into homodimers which activates the tyrosine kinase domain and downstream pathways. This signalling of EGFR is essential in skin re-construction. The activation of EGFR promoted the following cellular responses: migration, proliferation, cytoprotection and epithelial-mesenchymal transition [5].
Since EGF has multiples valuable biological functions, it has been used in the treatment of hard-to-heal wounds, including diabetic foot ulcers and bedsores [6-8]. EGF can also stimulate the production of collagen and elastin, which further extend its application on cosmeceutical use [9,10]. Though there are various applications of EGF, the commercial utilization of EGF is very low due to the high cost in extraction of adequate amount in natural hosts with only a low abundance of EGF.
Many groups have tried to produce EGF using the recombinant technology via different approaches, including synthesis, intracellular expression and secretion, in order to expand its application on medical or cosmeceutical industry [11-13]. However, only fusion protein may result because of the failure to remove signal peptides or affinity tags.
Our group has been concentrated on employing different inteins to produce various recombinant proteins, including but not limited to interleukin 3, interleukin 4 receptor, interleukin 6, stem cell factor (manuscript in preparation) and basic fibroblast growth factor as well as focusing on in vitro translation (IVT) system for expressing recombinant proteins [14]. Our group has been searching for different ways, especially inteins and IVT system, to produce soluble and human bio-identical EGF from bacterial or mammalian host. Some of the group also tried to employ intein approach in facilitating the expression of EGF, however, only insoluble EGF fusion proteins was obtained and protein refolding is needed during the purification of EGF [15]. The protein conformation may be incorrect during the refolding process and eventually greatly affect the bioactivity of the EGF [16]. In this study, we aimed to produce soluble EGF via intein mediated system by controlling the C-terminal cleavage of chosen intein or ideally by auto-cleavage without the need of protein refolding process. A GST tag was fused on the N-terminus of chosen intein, gp41-1, to facilitate the downstream purification and also, increase the solubility of EGF fusion protein. A strong T7 promoter was employed to initiate the transcription and improving the yield of soluble EGF fusion protein. The result was satisfactory under IPTG induction at low temperature. No significant change was found in upscaling the production of fusion from 1 L shake flask to 45 L fermentation. EGF was shown to be able to separate from the fusion protein under incubation with low concentration of DTT in natural pH by performing the on-column cleavage. The mitogenicity result also showed that the purified EGF was showed to be highly bioactive.
To explore the effectiveness of EGF, various concentration of EGF with the addition of bFGF was added into homemade aqueous cream in treating patients with different levels of skin ruptures. EGF was proven to be effective in treating patients with bedsores, skin injuries and diabetics in a short period. Our study may further enable and promote the use of EGF in both medical and cosmeceutical sector.
Bacterial strains and chemicals
E. coli strain, DH5α and T7 express, and restriction enzymes were purchased from New England Biolabs (Ipswich, MA). The synthetic DNA fragments and antibody against EGF were purchased from Thermo Fisher Scientific (Ipswich, MA). All other chemicals were purchased from Sigma- Aldrich (St. Louis, MO) unless otherwise specified.
Construction of EGF expression vector
The expression construct, pET42a (+)-GST-gp41-1-EGF, was constructed in the following way. A synthetic DNA fragment, encoding the sequence of NotI-stop codon-EGF-gp41-1-thrombin site-SpeI from 5’ to 3’ end, was synthesized by Thermo Fisher Scientific. The synthetic DNA fragment was amplified by PCR extension using the forward primer 5’- AAAAAGCGGCCGCTTAGCGCAGTTC-3’ and backward primer 5’- AAAAAACTAGTCTGGTGCCACGCGGTAGTT-3’. The amplified PCR product was purified by Axygen AxyPrep PCR Clean-Up Kit and digested with NotI and SpeI. The digested fragment was purified from 1% agarose and ligated into pET42a (+) digested with the same restriction enzymes. The plasmid sequence of pET42a (+)-GST-gp41-1-EGF was confirmed by Sanger sequencing.
Expression of EGF fusion protein in shake flask
The plasmid, pET42a (+)-GST-gp41-1-EGF, was transformed into the T7 Express. A single colony of transformant was grown at 37°C, (with rotations at 250 rpm) in 1 L LB medium supplemented with 40 μg ml-1 of kanamycin. When the A600 value reached 0.5, the growth temperature was reduced to 16°C and a final concentration of 0.1 mM IPTG was added. The culture was allowed to grow overnight. 1 ml cell pellet was collected and resuspended in 200 μl of resuspension buffer (50 mM Tris-Cl, 200 mM EDTA, pH 8.0) supplemented with 1x PMSF, aprotinin, benzamidine and leupeptin, followed by incubation on ice for 5 min. The mixture was then treated with 120 μl of lysozyme solution (1 mg mL-1) at 37°C for 20 min. 80 μl of lysis buffer (10 mM EDTA, 10% Triton X-100, and 50 mM Tris-Cl, pH 8.0) was then added. The tube with solutions was inverted gently, followed by centrifugation at 14,800 rpm for 5 min. The cell lysate samples were analyzed by Western blotting.
Up-scaling expression of EGF fusion protein in fermentor
The T7 Express, pET42a (+)-GST-gp41-1-EGF transformant was grown at 37°C, (with rotations at 250 rpm) in 1 L LB medium supplemented with 40 μg ml−1 of kanamycin. When the A600 value reached 1, the entire volume of culture was subculture into a 50 L fermentor containing 44 L LB medium. The culture was allowed to grow at 37°C, (with rotations at 100 rpm, 1.5 vvm) until A600 value reached 0.5, the growth temperature was reduced to 16°C and a final concentration of 0.1 mM IPTG was added. The culture was allowed to grow overnight with 1 M H2SO4 and 1 M NaOH in maintaining the pH at 7.0. Cell pellet was harvested by continuous centrifugation and washed with buffer A (1x PBS with 1x PMSF, aprotinin, benzamidine and leupeptin) two times prior to long term storage
Protein purification and inducted cleavage of EGF
The harvested cell pellet was resuspended in 400 ml buffer A. Resuspension was lysed by sonication (10s sonication with 30s intervals for 30x), and then centrifuged at 10,000 rpm for 30 min. Supernatants were clarified and loaded onto the glutathione agarose 4B resin column, followed by washing with buffer A for 10 bed volume. The induction buffer, buffer C (50 mM Tris–Cl, 1 mM EDTA, 300 mM NaCl, 2 mM DTT, pH 8) was added onto the column and allowed incubation at room temperature for 24 hours. Elution was done by adding 3 bed volumes of buffer C. Eluates were saved for Western blot analysis and dialyzed with 0.1x PBS before lyophilization for other purposes.
Biological assays of EGF
The mitogenic effects of reconstituted EGF on the proliferation of NIH/3T3 fibroblast cells were analyzed by the MTT assay [14-16].
Source of bFGF
Transfection, purification and bioassay of human basic FGF were done following Kwong et. al protocol as described previously [14-16].
Preparation of rejuvenating cream and directions
Different concentration of lyophilized EGF and bFGF [14], (1) 0.005% EGF, (2) 0.02% EGF and 0.00015% bFGF, (3) 0.04% EGF and 0.0003% bFGF, were mixed with homemade aqueous cream (aqua, phenoxyethanol, cetostearyl alcohol, ceteareth-20, liquid paraffin and white soft paraffin) for 10 mins. The mixed cream was topically applied to various wounds, including bedsore, wound and diabetic foot ulcers of different stages. All treatments were carried at patients’ home. Wounds included diabetic foot ulcers, bedsores, laceration, etc. The wounded area was cleaned completely following the Hong Kong Hospital Authority procedures. In brief, 1) clean the wound area thoroughly, 2) apply a small amount of cream to the disinfected area 3) apply sterile adhesive dressing. 4) monitor the healing process closely. Repeat step 1) to 4) until the wounds were fully healed. The aforesaid procedures are repeated twice daily.
From the results of the above case studies, the EGF purified from the fusion protein was proven to be highly active. Our findings prove that the final reconstituted EGF obtained by intein approach is bioactive in treating patients with different skin injuries, though the healing process may highly depends on the physiological conditions of the tested subject. More participants may be recruited for investigating the efficiency of EGF in treating different skin injuries in the near future to minimize the variables between different subjects.
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