Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation

Journal Pre-proof Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation

Lamis Al-Homsi, Hasan Naser El-Din, Hossam Murad, Abdul Qader Abbady PII:

S2452-0144(19)30138-4

DOI:

https://doi.org/10.1016/j.genrep.2019.100496

Reference:

GENREP 100496

To appear in:

Gene Reports

Received date:

29 July 2019

Revised date:

9 August 2019

Accepted date:

26 August 2019

Please cite this article as: L. Al-Homsi, H.N. El-Din, H. Murad, et al., Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation, Gene Reports (2018), https://doi.org/10.1016/ j.genrep.2019.100496

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© 2018 Published by Elsevier.

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Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation Authors names Lamis Al-Homsi1; Hasan Naser El-Din1; Hossam Murad2 and Abdul Qader Abbady2* Author affiliations: Department of Animal Biology, Faculty of Sciences, Damascus University, Syria.

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Division of Molecular Biomedicine, Department of Molecular Biology and Biotechnology, Atomic Energy

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Corresponding author. E-mail: [email protected]

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Commission of Syria (AECS), Damascus, Syria.

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Tel.: +963 11 213580; fax: +963 11 6112289.

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Lamis Al-Homsi: [email protected]

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Authors e-mails:

Hasan Naser El-Din: [email protected]

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Hossam murad: [email protected]

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Abdul Qader Abbady: [email protected]

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Abstract Background: Human insulin like growth factor-1 (IGF1) is a hormonal peptide associated with growth and development in mammalian. It has become a particularly attractive therapeutic target because of its role in various physiological processes. IGF1 binding to its receptor IGF1R on the surface of the cell triggers a signaling cascade leading to proliferative and anti-apoptotic events. Results: We constructed an adapter of codon optimized IGF1 gene with two different linkers;

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rigid: EAAAK (E) and flexible: GSGSG (G), which was cloned in the plasmid pRSET-sfGFP as a

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fusion partner to the superfolder form of green fluorescent protein (sfGFP), resulting in the

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constructs pRSET-sfGFP-IGF1-(E or G). The expressed 6× His tagged sfGFP-IGF1-E protein (36

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kDa) was purified from the cytoplasm of E. coli by metal affinity chromatography. Its purity was confirmed by SDS-PAGE blue staining and immunoblotting with anti-His or anti-GFP antibodies.

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Proper folding of sfGFP-IGF1-E was confirmed by indirect ELISA that revealed the ability of the

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sfGFP-IGF1-E but not the sfGFP, to bind to the immobilized IGF binding protein 3 (IGFBP3), and the ability of IGFBP3 to bind to the immobilized sfGFP-IGF1-E. Both interactions were able to be

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detected by anti-GFP and anti-IGFBP3, respectively. XTT test confirmed the bioactivity of sfGFP-

sfGFP-IGF1-E.

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IGF1-E after MCF-7 and HepG2 cancer cell lines treatment with increased concentrations of

Conclusions: The procedure described in this study facilitate the production of effectively sfGFP-IGF1-E fusion protein in sufficiently large amounts, which provides a promising curative compound for many disorders. Keywords: IGF1, sfGFP, gene cloning, protein expression,

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Highlights   

IGF1 gene was constructed and cloned in pRSET-sfGFP plasmid. Proper folding of sfGFP-IGF1 fusion protein was evaluated through binding with IGFBP3. sfGFP-IGF1fusion protein induces cell proliferation in MCF-7 and HepG2 cancer cells.

Background

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Insulin-like growth factor I (IGFI) is a small 70 aa polypeptide hormone with a molecular

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weight of 7.7 kDa. It shares >60% homology with IGF-II and by 50% homology with proinsulin

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structures (Arai et al., 2001; Hu et al., 2004; Philippou et al., 2014). IGF1 is produced primarily in the liver under the direct stimulation of Growth Hormone (GH). Although, virtually every tissue is

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able to secrete IGF1for autocrine/paracrine purposes. It is partly responsible for systemic GH

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activities although it possesses a wide number of own properties (anabolic, antioxidant, antiinflammatory and cytoprotective actions) (Puche and Castilla-Cortazar, 2012). IGF1is a relevant

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hormone both in embryological and postnatal states (Puche and Castilla-Cortazar, 2012). IGF axis

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is a highly conserved signaling pathway, with a crucial role in cellular and tissue regeneration

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through its proliferative and anti-apoptotic activities (Pollak et al., 2004). In addition, it is highly expressed and known to promote the growth of several tumors (Mazziotti et al., 2002). Most serum IGF1 is bound to IGFBP3; the most abundant protein in the circulation, where they form a ternary complex of 150-kDa with an acid-labile subunit (ALS). This bonding regulates the biological accessibility and activity of IGF1 by increasing the half-life of circulating IGF1 (from 12 minutes to 12 hours) and controlling its availability for receptor. IGFBP3 binds IGF1 with high affinity (Kd = 10-10 M) and facilitate the transfer of IGF1 into the extracellular fluid, which then reaches the target tissue (Rajaram et al., 1997).

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Journal Pre-proof An increasing list of studies has been reported emphasizing the role of this molecule in many different organs and systems. IGF1 helps combat autoimmunity by increasing T Regulatory Cells, and it decreases MHCI gene expression (Smith, 2010). IGF1 improves learning and memory in animal models (Lupien et al., 2003). It is important for building muscle, and for reducing muscle loss in aging and disease (Adams and McCue, 1998). In addition, IGF1 has shown to have antiinflammatory and anti-oxidant effects on blood vessels, stabilizing existing plaque and reducing additional plaque accumulation (Higashi et al., 2014). It is prescribed for children with IGF-

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deficiency (such as Laron syndrome) to help them restore height (Backeljauw et al., 2013). IGF1 is

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essential for normal fetal growth and development (Agrogiannis et al., 2014). Sharing homology with human insulin, IGF1 also can increase insulin sensitivity and possess hypoglycemic effects

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(Panahi et al., 2004). Therefore, it may become a substitute therapeutic agent to cure subjects with

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functional defects in their insulin receptor. With clinical significance in medicine, the development

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for high-efficient production of IGF1 is very demanding to fulfill its increasing market (Zhang et al., 2010).

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For all these benefits, it is better to have this molecule in an effective form to be tested for more

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studies. IGF1 considered as a small peptide and this kind of peptides have difficulties to be

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produced and monitored, so the principle reason for this study is to find a way to obtain IGF1 in an effective, high purity form and in sufficient amounts for more clinical trials. Gene fusion techniques allow the production of recombinant proteins displaying the properties of both fused proteins. Recently, new applications have emerged such as protein display, drug targeting and delivery, protein engineering and refolding (Uhlen et al., 1992; Chen et al., 2010). Various medical peptides have been produced using recombinant protein techniques in E. coli and yeast (Nicolas et al., 2011; Nielsen, 2013). Due to the importance of these medical peptides, they usually expressed as fusion with carrier proteins, a strategy necessary to save them from proteolytic degradation (Ryan and Henehan, 2013). Furthermore, the resulting fusion protein can 4

Journal Pre-proof obtain multiple functional properties derived from each of its components (Uhlen et al., 1992; Vai et al., 2000; Panahi et al., 2004; Al-Homsi et al., 2015). Linkers in fusion proteins are usually inserted to decrease the interaction especially at the protein folding stage (Arai et al., 2001; Hu et al., 2004). Generally, empirical linkers were classified into three types: flexible, rigid and cleavable linkers. Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. Generally, they are composed of small non-polar or polar amino acids (such as glycine-serine linker GSGSG), which can provide structure flexibility, improve protein

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stability, or increase biological activity (Wriggers et al., 2005; Amet et al., 2009). While rigid

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linkers consist of α-helix–forming peptide, such as (EAAAK) (Chen et al., 2013).Cleavable linkers are introduced to release free functional domains in vivo. This type of linker may reduce steric

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hindrance, improve bioactivity, or achieve independent actions/metabolism of individual domains

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of recombinant fusion proteins after linker cleavage (Chen et al., 2013).

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Both chemical and enzymatic cleavage have been used to get rid of the tags from fusion proteins (Arnau et al., 2006). Due to the specificity and stringency of the proteases (Parks et al.,

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1994), we used Tobacco etch virus (TEV) as a cleavable reagent, which is active on a variety of

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substrates and cleaves efficiently at low temperatures. TEV distinguish a specific sequence of

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amino acid ENLYFQ/G with high efficiency and cleaves between Q and G (Polayes et al., 1998; Kapust et al., 2002), which are included between the fused proteins. Recently, fluorescent protein is widely used as gene reporter and protein marker. Green fluorescent protein (GFP) has been used as a marker for many in vivo and in vitro applications due to GFP’s ability to fold and form a visually fluorescent chromophore through an internal posttranslational autocatalytic cyclization that does not require any cofactors or substrates (Shimomura et al., 1962). Due to the high thermodynamic stability, robust folding kinetics of GFP (Ormo et al., 1996). It has been used to study protein dynamics in living cells (Lippincott-Schwartz et al., 2001), in bacterial protein localization (Phillips, 2001), real time molecular and cellular 5

Journal Pre-proof analysis (Taylor et al., 2001) and as a reporter in protein folding (Waldo, 2003). Lately, a superfolder form of GFP was engineered by Waldo and coworkers (sfGFP), that is more resistant to denaturation and aggregation during refolding, and improved folding kinetics (Pedelacq et al., 2006; Andrews et al., 2007). In addition, the fusion of sfGFP increases the solubility of the expressed protein (Wu et al., 2009). sfGFP has improved protein tagging and detection both in vitro and in vivo using self-assembled sfGFP fragments. Furthermore, sfGFP cold fold independently of its fusion partner statues (Cabantous et al., 2005b; Abbady et al., 2014; Al-Homsi et al., 2015).

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The development of an efficient detection and purification procedure for IGF1 could greatly

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facilitate its uses as a curative compound for many disorders. We present here two different

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constructs for IGF1 gene (after codon optimization for E.coli protein expression system) with two linkers rigid and flexible. The two IGF1 constructs were fused with sfGFP as a fusion partner in the

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presence of TEV cutting site as a cleavable linker between IGF1 and sfGFP genes. sfGFP-IGF1-E

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recombinant fusion protein was purified, detected and tested for its proper folding and for its

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biological activity in different cancer cell lines.

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Methods

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Bacterial strains, growth conditions and plasmids E. coli strains TOP10 and BL21 (DE3) Gold were purchased from Invitrogen and Novagen, respectively. They were used in cloning and protein expression after transformation by electroporation with the plasmids pRSET (Invitrogen) and pRSET-sfGFP was prepared previously (Al-Homsi et al., 2012). For general maintenance and protein expression, E. coli were grown in Luria Broth (LB) (Bio Basic INC) with the required antibiotic at 100 mg/ml (Ampicillin; Applichem) at 37°C.

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Journal Pre-proof Cloning of IGF1 The DNA adapter containing IGF1 gene was synthesized using 12 overlapping primers and two additional amplification primers 20 mer (Supplementary material File S1). Primers were designed to add a short sequence between the fused proteins containing one of two different linkers; a flexible GSGSG referred to by the letter G and a rigid EAAAK referred to with the letter E, upstream a TEV protease-cutting site. Amplified DNA fragment has terminal restriction sites for the enzymes BamHI, HindIII, which are necessary for cloning in pRSET-sfGFP and pRSET plasmids.

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Designing of primers was based on the amino acid sequence of IGF1 from the genbank (Genbank

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Accession Number. NP_001104753) and on the codon preference of the BL21 host cell.

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A simple two-step Splicing by Overlap Extension (SOE) PCR method was done for the rapid construction and amplification of the DNA adapter containing IGF1 gene. In this reaction, 12

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overlapping oligonucleotides were used to synthesize the cloning adapter of IGF1 gene through 20

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sequential rounds of PCR. After this step, PCR product from the first reaction was used as a

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template DNA for a next amplification PCR (35 rounds) in which two specific primers were used (Adapter F/Adapter R). The first step of the method is to mix the overlapping oligonucleotides (IF-1

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– IF-6, IR-1 – IR-6), which were used as DNA template in the first PCR; The IR-1 primer was

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synthesized as IR-1E referred to liker EAAAK and IR-1G referred to linker GSGSG. The reaction included an initial denaturation for 3min at 95ºC then 20 cycles with incremental annealing temperature every 5 cycles (45ºC, 50ºC, 55ºC, 60ºC). This was done to maximize the chance of oligonucleotides hybridization necessary for DNA synthesis. Each PCR cycle included denaturation for 15sec at 94ºC then the incremental annealing for 30sec followed by extension for 45sec at 68ºC. Amplified adapter of IGF1 as well as the plasmids were digested with the restriction enzymes BamHI and HindIII. Digested product were then ligated using T4 DNA ligase (Fermentas). The new plasmid constructs pRSET-sfGFP-IGF1-E or G and pRSET-IGF1-E or G (Supplementary material figures S1, S2, S3 and S4) were transformed into electro-competent E. coli TOP10 cells by 7

Journal Pre-proof electroporation. Positive clones were confirmed by Colony PCR screening using plasmids specific primers. Then, they were grown in LB/ampicillin medium for plasmids isolation by Plasmid Miniprep Kit (Qiagen). DNA sequencing and digestion with restriction enzymes were performed to confirm successful cloning. Expression and purification of soluble sfGFP-IGF1 protein in E. coli Freshly prepared E. coli BL21 (DE3) Gold cells were used to transform the confirmed plasmid constructs by electroporation. For scale-up culture, the bacteria with positive plasmids were grown

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in LB medium using 250 ml shake flasks till an optical density of 0.5 to 0.7 was reached and then

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Isopropyl β-D-thiogalactopyranoside (IPTG, Promega) was added to a final concentration 0.3 mM

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for 16 h at three different temperatures 19º, 28º, 37ºC. After induction, the bacterial cells were

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harvested by centrifugation at 8000 rpm for 8 min at 4˚C. Cell pellets were suspended in PBS 1×, then lysed by sonication (Lab Sonic) on ice and the lysate was centrifuged at 8,000 rpm for 20 min

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at 4°C. The clear supernatant was loaded onto 5 ml column of Nickel charged Nitrilotriacetic acid

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(NTA) superflow Sepharose (Qiagen) using fast protein liquid chromatography (FPLC) AKTAprime plus system (GE lifescience). After washing, sfGFP-IGF1-E protein was eluted from

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the column with elution buffer containing 500 mM imidazole. After elution, the pure protein was

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concentrated on Vivaspin concentrators with a molecular mass cutoff of 10 kDa (Vivascience). The purity of sfGFP-IGF1-E was assessed using a Coomassie-stained SDS-PAGE. Then, bradford method was applied for determining concentration of the purified protein. In order to view real-time expression and purification of sfGFP-IGF1-E, the fluorescence of 100 μL sample from each step of the procedure was monitored by measuring the fluorescence density, expressed as a relative fluorescent unit (RFU), at excitation wavelength 485 nm with emission wavelength 538 nm (Fluoroskan Ascent FL, Thermo Labsystems).

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Journal Pre-proof Immuno-blotting and coomassie blue staining of SDS–PAGE Protein samples were separated by SDS-PAGE using a Bio-Rad mini-Protean II system following the manufacturer’s instructions. Gels were prepared using stacking gel 5% and running gel 15%. After electrophoresis, the gel was stained in coomassie blue for 2 h then distained in 5% acetic acid, 10% methanol. For immuno-blotting, the proteins were separated on SDS-PAGE before it was blotted onto 0.45 µm nitrocellulose membranes (BioRad) using 1× blotting buffer (25 mM Tris-base, 200 mM

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glycine, 0.1% SDS and 20% methanol). After incubation in 5× blocking buffer, first antibody

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(mouse-anti-IGF1 (1:500)) were added for 1 h at room temperature. After several washes with TBS-

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T, second conjugated antibody (goat-anti-rabbit-AP (1:2000)) was added for 1 h at room temperature. Finally, Nitro blue tetrazolium chloride (NBT, 0.05%)/ 5-bromo-4-chloro-3-indolyl

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phosphate (BCIP, 0.025%) (Sigma) substrate was added in AP buffer (100 mM Tris-base, 100 mM

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NaCl, 5 mM MgCl2, pH 9.5) for revealing bands.

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Indirect ELISA assay for testing sfGFP-IGF1-E and detection

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Indirect ELIZA was performed using Maxisorb 96-well plates (Nunc). For sfGFP-IGF1-E detection, the plate was coated with antigens; sfGFP, sfGFP-IGF1-E and commercial IGF1 prepared

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at 2.5 µg/ml. And for protein-protein interaction, it was coated either with IGFBP3 30 nM (Sigma) or with IGF1 (Sigma) and sfGFP-IGF1-E 100 nM, by overnight incubation (at 4ºC) with 100 µl/well prepared in carbonate buffer. Residual protein binding sites in the wells were blocked for 1 h at 37ºC with 5× blocking buffer (3% skimmed milk and 1% BSA) in TBS-T (20 mM Tris-base, 150 mM NaCl, 0.05% Tween-20, pH 7.5). For protein-protein interaction, next antigens; sfGFP, sfGFPIGF1-E, IGF1 were prepared at 100nM and added or IGFBP3 at 30 nM. Dilutions of primary antibodies (anti-IGF1, anti-GFP, anti-His, anti-IGFBP3) were prepared and added to the wells according to what was indicated in each experiment. After several washes with TBS-T, detection 9

Journal Pre-proof was done with diluted (1:2000) goat anti-mouse or goat-anti-rabbit IgGs conjugated to horseradish peroxidase (HRP, Bethyl laboratories). The absorption at 450-nm was measured after adding peroxidase substrate 3,30,5,50-tetramethylbenzidine (TMB; Sigma) followed by stopping buffer (1 M of H2SO4) to terminate the enzymatic reaction of peroxidase. Cleavage of sfGFP-IGF1-E fusion protein by TEV proteases The purified sfGFP-IGF1-E and TEV protease were used in the cleaving reaction. Protease digestion was performed in a mass ratio of substrate to enzyme 100:4 (w:w) in a reaction buffer

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(75mM NaCl, 0.5mM EDTA, 25mM Tris pH 8, 10% glycerol and 100mM DTT) at 30°C for 16h.

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Cell culture and cell proliferation assay

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The extent of cleavage of the samples was determined or compared by SDS−PAGE analysis.

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HepG2 human hepatocarcinoma cells and MCF-7 human breast cancer cells were purchased

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from Sigma-Aldrish, then were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 50 U/ml penicillin/streptomycin and 2 mM L-glutamine. The cells were cultured at 37˚C in

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5% CO2. All materials used in the cell culture were supplied by Gibco-BRL. Before experiment,

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cells were seeded at a density of 104 in a 96-well plate. After a 24 h of culture, cells were treated with increased concentrations of sfGFP-IGF1-E at 0, 10, 100, 1000 ng/ml and incubated for 48h.

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Cell viability was counted using an XTT assay kit (Roche) following the manufacturer's instructions. The number of living cells was quantified by measuring absorbance at a wavelength of 450 nm using a microplate reader (Multiskan EX Microplate Readers; Thermo Scientific) and the absorption of the control cells was set to 1. The graph of cell viability was demonstrated as fold change in viability from the untreated MCF-7 and HepG2 cells. The growth of the sfGFP-IGF1-E treated cells was compared with the growth of untreated cells. Treatment with each sfGFP-IGF1-E concentration was performed in four replicate.

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Results Cloning of IGF1 gene into pRSET and pRSET-sfGFP plasmids T7 promoter technology is widely used for high throughput protein expression in E. coli strains carrying the lambda DE3 lysogen (Studier and Moffatt, 1986; Studier et al., 1990). The plasmid pRSET-sfGFP (Al-Homsi et al., 2012) is a T7 promoter-dependent system which produces cytosolic N-terminal 6×His-tagged proteins in E. coli cells. Tagging of recombinant proteins is indispensable

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for the subsequent steps of purification and detection. The adapter of IGF1 was designed using 12 overlapping primers (IF-1 – IF-6, IR-1 – IR-6), the primer IR-1 includes the sequence of linker

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either E or G which added to the adapter (Fig. 1A). Then it was amplified by two specific primers

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(Adapter F, Adapter R) resulting in the amplification of a single DNA fragment of 317 bp (Fig 1B).

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Digestion of this DNA fragment resulted in a sticky ended fragment ready to be ligated in the

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pRSET-sfGFP and pRSET plasmids after being linearized with the two restriction enzymes BamHI, HindIII. Digested pRSET-sfGFP and pRSET plasmids as well as the two adapters of IGF1-E or G

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insert were purified from gel and used in a ligation reaction at 1:3 ratio.

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Fig 1. Cloning of IGF1 into pRSET-sfGFP and pRSET plasmids

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A) Schematic illustration of the IGF1 adapter construction using overlapping primers. (B) Agarose gel electrophoresis of PCR products of 317 bp using (IF-1 – IF-6, IR-1 – IR-6) then (Adapter F, Adapter R) (lane 1 for IGF1-E) (lane 2 for IGF1-G). Results of colony PCR screening performed on 3 randomly selected clones after E. coli TOP10 transformation with the ligation reaction products. Positive clones, which contain full-length IGF1-(E or G) (C) and sfGFP-IGF1-(E or G) (D) genes, were indicated (+). DNA fragments were separated into 1.5 % agarose gel where side arrows indicate the expected positions and sizes (in bp) of these fragments as well as the bands of the DNA molecular weight marker (M) of size markers (M).

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Ligated products were used to transform the E. coli TOP10 cells by electric shock and after

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short time for cells to recover; positive colonies on the plates were screened by PCR using pRSETsfGFP and pRSET specific primers(GFP-F/TR) and (TF/TR) respectively (Supplementary material file S1). This approach enabled the distinguishing between two types of colonies; empty (pRSETsfGFP and pRSET) containing colonies which resulted in small amplified DNA fragment (155 bp and 295 bp, respectively). And (pRSET-sfGFP-IGF1-E/G and pRSET-IGF1-E/G) containing colonies which gave a big fragment of (377 bp and 518 bp, respectively) due to the presence of the insert gene within (Fig. 1D – 1C). Positive colonies were grown and used for plasmid minipreparation and the right structure of the plasmid constructs were verified by digestion with the same restriction enzymes used in the cloning and by sequencing. 12

Journal Pre-proof Expression and purification of recombinant proteins The IGF1 is a relatively small peptide of 8 kDa after codon optimization was fused with Nterminal 6× His-tagged sfGFP of 27 kDa (Fig 2A) resulting in the fusion protein sfGFP-IGF1-(E or G) (Fig 2B-2C). The production of sfGFP-IGF1-(E or G) and IGF1-(E or G) proteins were obtained after transformation of E. coli BL21 (DE3) Gold cells with the confirmed plasmid constructs. Cells were grown in liquid LB medium supplemented with antibiotic and protein expression was then induced by IPTG at 19º, 28º, 37 ºC. Protein extraction from cytoplasm or inclusion bodies were

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followed by Dot blot then detected by rabbit anti-GFP antibody. As shown in (Fig 3A) soluble

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protein was obtained by lowering temperature, while at 28ºC and 37 ºC the expressed proteins were

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accumulated in inclusion bodies. However, the construct pRSET-sfGFP-IGF1-G has low protein

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expression level comparing to pRSET-sfGFP-IGF1-E.

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Fig 2. Designing of the sfGFP-IGF1 fusion protein

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(A) Schematic representation of the IGF1 adapter and the two recombinant proteins; sfGFP-IGF1-(E or G) and sfGFP, used in this study. The theoretical molecular size (kDa) and molecular weight (pMoles/μg) are shown to the right of each recombinant construct. Positions of the different elements; 6 × His tag, GSSSG and EAAAK linkers, and TEV cutting site, are indicated using specific symbols *, ●, and ♦, respectively. (B) Cartoon representation of the modelled 3D structure of the sfGFP-IGF1-E fusion. (C) Cartoon representation of the modelled 3D structure of the sfGFP-IGF1-G fusion, where TEV cutting site, GSSSG and EAAAK linkers and the N-terminal 6 × His tag are shown. Structure simulation was predicted using Phyre2 server (Kelley et al., 2015).

Purification of the fusion protein sfGFP-IGF1-E from cytoplasmic extract was done on immobilized-metal affinity chromatography, using Nickel-charged NTA column installed on AKTAprime system. The UV-detector, supplemented with this system, enabled the real-time monitoring of the different steps of sfGFP-IGF1-E purification (Fig. 3C). The protein expression and purification procedures of sfGFP-IGF1-E were followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) stained with coomassie brilliant blue staining

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Journal Pre-proof (Fig. 3B). Moreover, it was immune blotted then detected by rabbit anti-His antibody. However, no

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distinct expression of the construct pRSET-IGF1 in cytoplasm or in inclusion bodies (Fig. 3D).

Fig 3. Expression and purification of sfGFP-IGF1-E

(A) Dot blot of sfGFP-IGF1-(E or G) was done after protein expression in shaking flask using different temperature for induction 19˚C, 28˚C, 37˚C, protein extraction was prepared from cytoplasm and inclusion bodies. Cytoplasmic extract of sfGFP-IGF1-(E or G) (lane 1, 3 respectively), inclusion bodies extract of sfGFP-IGF1-(E or G) (lane 2, 4 respectively). All samples were detected by rabbitanti-GFP. (B) Protein migration in SDS-PAGE (acrylamide 15 %) of the protein samples obtained after different steps of expression and purification; total cytoplasmic extract after 16 h of IPTG induction (lane 1), flow-through sample from Ni-NTA column (lane 2) and purified sfGFP-IGF1-E protein (lane 3). (C) Diagram of purification procedure using Ni+-NTA column installed on FPLC AKTAprime system. Continuous line represents the absorbance of the eluate, different purification steps are shown below and peaks of the flow-through sample and of purified sfGFP-IGF1-E are indicated. Dashed line represents conductivity of the eluate. (D) Detection of the purified sfGFP-IGF1 was done after SDSPAGE separation by immune blotting using Rabbit-anti-His, total cytoplasmic extract (lane 1), pure sfGFP-IGF1-E (lane 2), detection of pRSET-IGF1 expression in total cytoplasmic extract (lane3), or in inclusion bodies (lane 4), control GFP (lane 5).

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Journal Pre-proof A remarkable expression of sfGFP-IGF1-E could be observed after IPTG induction followed with 16 h of incubation at 19 C. Although, the expressed protein was totally purified from bacterial cytoplasmic extract by column purification, which yielded 90 % pure sfGFP-IGF1-E. The yield of purified recombinant protein estimably reached 150-200 mg/liter of bacteria culture. Evaluation and cleavage of purified sfGFP-IGF1-E In order to evaluate the proper folding of the purified recombinant protein against its specific antibody. Indirect ELISA was performed in sfGFP-IGF1-E, sfGFP and IGF1 commercial, precoated

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wells and the bound antibodies (mouse-anti-GFP, anti-His and anti-IGF1) were detected with a

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goat-anti-mouse secondary antibody conjugated to HRP (Fig. 4). Which revealed the detection of

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sfGFP-IGF1-E by the three antibodies, moreover sfGFP-IGF1-E binds anti-IGF1 with high affinity

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similar to IGF1 commercial.

Fig 4. Evaluation of the purified sfGFP-IGF1-E by direct ELIZA Chart for ELIZA test in the absence of antibodies (No Ab) or in the presence of anti-IGF1 (1:500) and anti-GFP (1:5000) and anti-His (1:3000) against immobilized sfGFP, sfGFP-IGF1-E and commercial IGF1 (0.25 µg/well of each antigen) or in the absence of antigen (No Ag).

For cleavage reaction, the mass ratio of the substrates sfGFP-IGF1-E and sfGFP-GH (as a control); which contain the protease recognition site, to the TEV protease enzyme was 100:4, so that 25 mg of the substrates were incubated with 1mg of the enzyme in the appropriate buffer for 16

Journal Pre-proof 16h 30°C. Protein samples from the cleavage reaction were prepared and then loaded onto 15% SDS-PAGE gel for electrophoresis, which was finally visualized by coomassie brilliant blue staining (Fig 5B). In this result, liberated GH (22 kDa) could be observed after the cleavage reaction

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(Lane 3). In comparison with uncleaved sfGFP-IGF1-E (Lane 5).

Fig 5. Characterization of sfGFP-IGF1-E fluuorescence and cleavage

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(A) The quantification of sfGFP fluorescence during sfGFP-IGF1-E expression and purification. The fluorescence was collected using fluoroskan reader with 485 nm excitation filter and 535 nm emission filter. (−): uninduced cell lysate; (+): induced whole cell lysate; S↑: supernatant of the sonication; F.T.: flow through fraction from Ni-NTA chromatography, E.: eluated fraction by imidazole from Ni-NTA chromatography. (B) SDS-PAGE (acrylamide 15 %) of protein samples obtained from cleavage reaction. TEV protease (lane 1), sfGFP-GH before cleavage (lane 2), GH and sfGFP were shown separated after cleavage (lane 3), sfGFP-IGF1-E before cleavage (lane 4), no separation of sfGFP-IGF1E components was detected after cleavage (lane 5).

Protein –protein interaction between sfGFP-IGF1-E and IGFBP3 Indirect ELISA assay was performed to monitor the interaction between IGFBP3 and IGF1 in free or fusion form (Fig 6). The results showed that immobilized IGF1 and sfGFP-IGF1-E 100nM were able to bind IGFBP3 30nM with high affinity, this interaction was detected by anti-IGFBP3 17

Journal Pre-proof (Fig 6A). Conversely, the binding of sfGFP-IGF1-E 100nM to immobilized IGFBP3 was detected by anti GFP (Fig 6B). While no significant signal was shown for the binding between immobilized

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IGFBP3 and IGF1 in the free form, which was detected by anti IGF1.

Fig 6. Testing the interaction of IGFBP3 with sfGFP-IGF1-E, IGF1 and sfGFP by ELISA (A) Indirect ELISA was performed using immobilized sfGFP-IGF1-E (100 nM), sfGFP (100 nM) and IGF1 (100 nM) after adding IGFBP3 (30 nM to the wells and the interaction was detected by a polyclonal anti-IGFBP3 (1:500). (B) Indirect ELISA was performed using immobilized IGFBP3 (30 nM). sfGFP-IGF1-E (100 nM), sfGFP (100 nM) and IGF1 (100 nM) were added to the wells and the interaction was detected using anti-GFP and anti-IGF1. (C) Optimal concentration of the sfGFP-IGF1-E and IGF1 for the interaction with IGFBP3 was determined by indirect ELISA using serial logarithmic concentrations (nM) of sfGFP-IGF1-E and IGF1. The detection of bound sfGFP-IGF1-E and IGF1 in ELISA was performed using rabbit anti-GFP, and mouse anti-IGF1, both rabbit and mouse sera were detected with a goat anti-rabbit-HRP and goat anti-mouse-HRP. The logarithmic fit equation and the accuracy (R2) are shown next to each curve

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Journal Pre-proof Testing the biological activity of the purified sfGFP-IGF1 on cancer cell lines MCF-7 and HepG2 cells were treated with increased concentration of sfGFP-IGF1-E (0, 10, 100, 1000 ng/ml) then incubated for 48h. In addition, MCF-7 was treated with 100 ng of commercial IGF1 (data not shown). The results showed increasing in cells proliferation of the treated cells comparing to untreated cells in concentration dependent manner. As shown in Fig 7, sfGFP-IGF1-E treated MCF-7 cells showed significant increasing in proliferation about 2.5 to 4.2 fold from control comparing with IGF1 treated MCF-7 cells that showed 2.5 fold from control.

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Also, treated HepG2 cells showed increasing in cells proliferation about 6.2 to 8.3 fold from

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control. We concluded that HepG2 cells could be more sensitive to sfGFP-IGF1-E treatment than

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MCF-7 cells, moreover, IGF1 commercial and sfGFP-IGF1-E were almost the same in biological

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activity.

Fig 7. Proliferative effect of sfGFP-IGF1-E on cancer cell lines Pro-proliferative effect of sfGFP-IGF1-E in a concentration-dependent manner, were studied in HepG2 and MCF-7 cells in comparison with control cells. Cell viability was measured by XTT assay. Values represent means ± SEM of four independent experiments

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Discussion This work described the establishment of a high-level expression system in E. coli using fusion protein technology, which represents one of the best solutions to achieve rapid, efficient, and costeffective protein expression and purification of recombinant proteins (Southward and Surette, 2002). It can facilitate purification, enhance protein expression and solubility, chaperone proper folding and reduce protein degradation or toxic effects (Davis et al., 1999). Up to now, a wide range of expression systems have been reported to produce native IGF1 such as yeast (Gill et al., 1999;

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Vai et al., 2000), cell-free system (Swartz, 2006), transgenic plants, and rabbits (Brem et al., 1994;

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Zinovieva et al., 1998; Panahi et al., 2004) whereas, it is found that E. coli host is known as more

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powerful expression system for recombinant production of native hIGF1 protein (Chung et al.,

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2000; Zhang et al., 2010; Ranjbari et al., 2015). Generally, E. coli expression systems are the most widely used host to produce industrial and pharmaceutical heterologous proteins owing to rapid

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growth, high cell density, high productivity, and low costs (Sorensen and Mortensen, 2005).

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Here we present the construction of IGF1 adapter that was codon optimized, to be more adequate with E.coli expression system, and then it was fused with sfGFP. This superfolder form of

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GFP has proven to enhance protein expression, detection and tagging both in vivo and in vitro with

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remarkable increase in fluorescence and solubility (Cabantous et al., 2005a; Wu et al., 2009). In addition, sfGFP was used in the expression of hard−to−fold proteins (Vasiljevic et al., 2006; Murad et al., 2014), toxins (Soler-Jover et al., 2004), proteases (Wu et al., 2009) and medical short peptides (Skosyrev et al., 2003; Al-Homsi et al., 2015), that tend to precipitate and accumulate in inclusion bodies, in both prokaryotic and eukaryotic cell types (Pedelacq et al., 2006). Visibility of GFP is the most distinguishable element from other fusion tags (Müller-Taubenberger and Anderson, 2007). Its unique green color (either visible blue light or UV light) allows us to monitor the existence of GFP fusion protein through purification steps even without any measurement apparatus like a UV spectrophotometer (Lee, 2009). (Fig 4 A) showed that the expression and purification procedure of 20

Journal Pre-proof sfGFP-tagged fusion protein can be monitored and quantified in real-time by the fluorescence emitted from sfGFP, thus greatly simplified the procedure of sfGFP tagged target proteins expression and purification (Wu et al., 2009). Theoretically, the function of a fusion protein would express both fused proteins function; however, their rolls and activity have been reported to be changed (Geng et al., 2006; Tranchant et al., 2006), or lost, and in some cases, the proteins were not even expressed (Doi and Yanagawa, 1999; Hong et al., 2006). Only a few proteins synthesized by fusion have exhibited the expected

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function (Lu et al., 2006; Orita et al., 2007). Generally, it is realized that closer spatial distance

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between the fused proteins affects their folding, function, and expression (Yang et al., 2015).

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Linkers are introduced to give the fusion protein a chance to achieve optimal biological activity and preferred structures. In this work, we produced two constructs using two linkers; flexible and rigid,

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in the presence of cleavable sequence to separate the fused protein. We used "GSGSG" as a flexible

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linker, which connect the functional domains passively and permitting certain degree of movement. However, this type of linkers may cause poor expression yields or loss of biological activity (Amet

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et al., 2009). As we reported here, low expression yield of the construct pRSET-sfGFP-IGF1-G in

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cytoplasmic extract and inclusion bodies after protein induction was confirmed by dot blot (Figure

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3A). In comparison with rigid linker, which was applied to keep a fixed distance between fusion proteins and to maintain their independent functions. We used (EAAAK) as a rigid linker (Arai et al., 2001). This linker displayed α-helical conformation, which was stabilized by the Glu− -Lys+ salt bridges within segments. The α-helical structure was rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone (Chen et al., 2013) sfGFP-IGF1-E protein was highly expressed using pRSET plasmid implemented with a T7 promoter. The optimal temperature to carry on expression was optimized for production process. Temperature is an important factor that affects plasmid stability and consequently the yield of protein production in culture. High temperature (37 oC) is suitable for high dry cell weight and high 21

Journal Pre-proof production (Huang et al., 2007; Ranjbari et al., 2015), however almost all the expressed sfGFPIGF1-E protein accumulated in the inclusion bodies. Generally, lowering temperature (19 oC) during gene induction lead to improve the quality and folding of the sfGFP-IGF1-E protein. We also constructed the control expression vector for IGF1 without any tags, but there was no detectable IGF1 under the same induction condition. Therefore, the fusion of sfGFP to IGF1 significantly increase the soluble production of IGF1. Also, it helps IGF1 to get the proper folding by forming its disulfide bridges.

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In addition, this plasmid provides high yields expression of N-terminal 6×His tagged sfGFP-

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IGF1 which could be efficiently purified using nickel charged resin. An important production yield

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of pure sfGFP-IGF1-E was obtained using this system, estimated to be about 150-200mg/L of bacterial culture, and the manufacturing cost was considerably lower, since most of the necessary

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elements, such as the expression plasmid pRSET-sfGFP, TEV protease and anti-GFP antibody,

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were prepared "in house".

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Cleavage of the fusion protein usually is necessary because of the possible interference with the structural or functional properties of the recombinant target protein. TEV protease represent the best

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choice for our system because of its high specificity and activity towards its cutting site. However,

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TEV protease failed to cleave our sfGFP-IGF1-E in comparison with the control sfGFP-GH, which was tested in previous work (Abbady et al., 2014). IGF1 is considered a small polypeptide with 7.7 kDa, it was fused with sfGFP which is 28 kDa. While GH is approximately in the same molecular weight of sfGFP. Differences in size might be the reason for TEV protease failing to cleave sfGFPIGF1-E. It would be of great interest to test more conditions in the future to optimize the optimal conditions for TEV cleavage. Pure sfGFP-IGF1-E was tested for its proper folding and for biological activity in order to confirm its ability to bind its specific antibodies, and cell surface receptor in order to activate signalling pathway leading to cell proliferating. Firstly, pure sfGFP-IGF1-E was able to bind its 22

Journal Pre-proof specific antibodies with high affinity, since we could use anti- sfGFP, anti-His and anti-IGF1 antibodies for detection in ELISA or immune blotting that confirm the proper folding of our fusion protein. However, mouse-anti-IGF1 antibody was not able to detect IGF1 in the free or fusion form in western blot. In addition, pure sfGFP-IGF1-E was tested for its ability to bind IGFBP3 in comparison with the commercial IGF1. Polyclonal anti-IGFBP3 was able to detect the binding between IGFBP3 and immobilized IGF1, in the fusion form (sfGFP-IGF1-E), showing the same affinity for IGF1 in its free form (commercial) (Fig 6A), contrariwise monoclonal anti-IGF1 failed

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to detect this binding in the opposite arrangement (Fig 6B); immobilized IGFBP3 bound to IGF1 in

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free or fusion form. Maybe differences in size between IGF1 and IGFBP3 in the complex IGF1IGFBP3 hide the unique epitope recognized by monoclonal anti-IGF1. However, in the fusion form

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sfGFP-IGF1-E we get the benefit of the tag sfGFP to detect this binding using polyclonal anti-GFP.

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Secondly, the results showed that the proliferation of the breast MCF-7 and liver HepG2 cancer

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cells has increased after treatment with pure sfGFP-IGF1-E in concentration dependent manner. The pro-proliferative action of IGF1 has previously revealed in MCF-7 cells through regulation of

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matrix metalloproteinase activity (MMP) and the invasive potential of MCF-7 cells (Walsh and

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Damjanovski, 2011) and in T47D breast cancer cells also as a mitogenic agents (halla et al., 2000).

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And it stimulates tumor growth in mouse model of colon cancer (Wu et al., 2002). In addition, IGF1 induces epithelial-mesenchymal transition (EMT) in HepG2 cells (Zhao et al., 2017)

Conclusion In conclusion, full length of IGF1 was constructed using overlapping PCR after codon optimization and adding a suitable linker to be fused with sfGFP. sfGFP-IGF1-E fusion protein was produced using an efficient E. coli protein expression system. After affinity purification, proper folding of sfGFP-IGF1-E was detected by ELISA and western blot. In addition, we confirmed the

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Journal Pre-proof ability of sfGFP-IGF1-E to bind IGFBP3 with high affinity using indirect ELISA. XTT method on MCF-7 and HepG2 treated cells revealed the pro-proliferative action of sfGFP-IGF1-E.

List of abbreviations IGF1, Insulin like growth factor; sfGFP, superfolder green fluorescent protein; IGFBP3, Insulin like growth factor binding protein; TEV, tobacco etch virus, AP, alkaline phosphatase.

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Ethics approval and consent to participate

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Declaration

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“Not Applicable”

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Consent for publication

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“Not Applicable”

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Availability of data and materials

additional file(s)).

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The data set(s) supporting the results of this articles is(are) included within the article (and its

Competing interests The authors declare that they have no competing interests.

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Authors’ contributions LH, carried out all experimental work. IGF1, designed sfGFP-IGF1-E bioactivity experiments, and wrote the manuscript. HND, read and commented the manuscript. HM, read and commented the manuscript. AA, led the work, designed all experiments, and commented the manuscript. All authors read and approved the final manuscript.

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Acknowledgements The authors would like to thank the Director General of the Atomic Energy Commission of

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Syria and the head of the Molecular Biology and Biotechnology department for their continuous

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support throughout this work

Funding

References

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This work was performed without any external funding.

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