THE EXPRESSION AND PURIFICATION OF OCTA-ARGININE APOPTIN AND ITS ABILITY TO KILL CANCER CELLS

Objective: In this research, chicken anemia virus apoptin optimized genetically for expression in Escherichia coli and also modified using (His)6 tag, (Arg)8 Methods: The modified apoptin gene was optimized using an Integrated DNA Technology (IDT). The gene (606 bp) then ordered and synthesized by Eurofins. The apoptin gene was expressed using E. coli BL21 CodonPlus as host, in cultivation temperature of 37 °C, and 25 °C and purified using Ni-NTA agarose beads. The addition of (His) tag, and HlyA tag intended for purification needs, penetration enhancement, and secretion from bacterial host to the growth media.


INTRODUCTION
Cancer is a term usually used to represent a group of diseases which affect various parts of the body. According to WHO, cancer is defined as the production of abnormal cells which is able to invade another part of the body and spread to other organs. This process is called metastasis, the largest cause of death by cancer. Based on WHO statistical data, cancer is one of the most deadly diseases in the world, with 8, 2 millions of deaths out of 14 million of cases in 2012. More specifically in South East Asia, WHO data showed 1, 72 million cases with 1, 17 million of deaths. As a developing country, Indonesia also has to face cancer problem. In Indonesia, cancer is ranked as the sixth most fatal disease. The most common cancer case in Indonesia are cervical and breast cancer where about 170-190 new cases are predicted to be found every 100,000 people [1].
A lot of methods have been developed to treat cancer. Generally, a treatment method for cancer can be classified into 6 groups, including chemotherapy, immunotherapy, radiotherapy, targeted therapy and transplantation. The most common cancer treatments in Indonesia consist of radiotherapy (70%), medical operation (20-25%) and chemotherapy (5-10%) [1]. According to National Cancer Institute, National Institute of Health United States, radiotherapy treatment uses high-energy radiation to kill cancer cells. Unfortunately, this method does not target cancer cells specifically. In turn, it would also harm normal cells and cause several side effects such as fibrosis, memory loss, and impotency.
In order to overcome those problems, cancer treatment which specifically target cancer cells and leave the normal cells unharmed is being developed. One of the potential candidates is the use of apoptin as a therapeutic protein. Apoptin consist of 121 amino acids and is known to be able to induce apoptosis specifically in cancer cells [2]. This mechanism is related to the specific kinase which is only available in cancer cells [2]. Apoptin production in bacterial host has been conducted by using various modifications, such as (His) 12 tag and (Arg) 8 tag [3]. The presence of related tags enables the protein purification process using chromatography column to be done in an easier way. On the other hand, the presence of related tags might also affect bioactivity of apoptin itself. It has been known that (his) 6 tag at C-terminus of heparin cofactor increases protein activity [3]. On the other hand, (his) 10 and (his) 7 Regardless of its modification, protein expression in a bacterial host might fail due to the differences of protein expression system between the original host and the new bacterial host. One of the approaches to increase protein expression in a bacterial host is codon optimization. Codon optimization has been successfully done to increase the expression level of halohydrin dehalogenase from Agrobacterium radiobacter in E. coli [5].
at N-terminus of tumor necrosis factor alpha decrease its activity drastically [4]. Hence, tag removal is considered necessary after protein purification in order to maintain the protein activity.
In this research, apoptin was modified in order to secrete the protein of interest, enhance its affinity for nickel chromatography column, and optimize the codon to be expressed in Escherichia coli. The recombinant protein was monitored their ability to kill cancer cells by MTT assay to HeLa and Widr Cells.

Gene construction
The information of apoptin gene was taken from gene bank with code AY171617.1. Some tags added into the apoptin sequence to gain protein. The modified apoptin gene was optimized using an online application from integrated DNA technology (IDT) accessed at http://sg.idtdna.com/CodonOpt. The gene (606 bp) then ordered and produced synthetically by Euro fins. The gene of interest placed inside pTAKN2 (3345 bp) and then moved from that plasmid into a new vector chosen, pET9a (4341 bp). The cut and ligate method used by NdeI and BamHI sites. The gene construction confirmed by agarose electrophoresis 0, 8 % (w/v) and DNA sequencing. DNA Sequencing primer was designed using Gene Runner.

Gene expression
The gene which has been constructed then transformed into and expressed using Escherichia coli. The expression is done using temperature variation (25 °C, 37 °C), and host cell variation (E. coli DH5a, BL21 pLysS, BL21 CP). The expression was done using Terrific Broth Hi-Media for 4 hours of cultivation and 4 hours of induction using IPTG 1 mmol.

Protein analysis
The protein analyzed using SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Gel concentration used are 15%. The protein from supernatant fraction of the cell culture precipitated by acetone precipitation method [6]. 250 µl of the supernatant mixed with 1 ml cold acetone, incubated for 2 h in-20 °C then centrifuged 12,000 rpm for 10 min. The pellets suspended in 1 time of phosphate saline buffer.

Detection of the inhibition of HeLa and widr cell by the MTT assay
The MTT method was used to study the effect of Apoptin 8 Arg on the growth of cancer cells [7]. HeLa and Widr cells were used for this assay. The viability of cell was analyzed when the cells incubated in the variation concentration protein for 48 h for HeLa cell and 72 h for Widr cell.

Gene construction
The gene construction confirmed using agarose gel electrophoresis and the result shown in fig. 1. The sequencing result of the gene constructed gave an exactly identic apoptin gene sequence as ordered to Eurofins. There are 3 N (could be any bases) read in the apoptin gene from Apoptin_R primer, confirmed to be the correct base by the reading from T7 Fw primer.  fig. 3 and fig. 4. Purified protein seen in SDS-PAGE of apoptin lysate, but not seen in pET9a lysate.
The expression also includes the supernatant fraction. If the protein designed successful, the protein should be secreted into the medium because of the HlyA tag. The precipitated protein from supernatant fraction analyzed by SDS-PAGE. Unfortunately, the supernatant fraction do not show a significant protein band around 20 kDa. The purification result shows that his-tagged apoptin successfully expressed. However, it's not significantly overexpressed in the cells. This might happen due to the secretion of protein accompanied by HlyA-tag or not optimal expression condition. The expression result shows that even without overexpression, his-tagged apoptin can be purified from BL21 pLysS lysate. Compared with BL21 pLysS, the purification and expression result of apoptin in BL21 CP is qualitatively better. On the other hand, a different result was reported by Lee et. al which shown that expression of modified apoptin in BL21 pLysS is better than BL21 CP [8]. This might happen due to different DNA construction for protein expression. Compared with that construction, the current research DNA construction uses His-tag instead of GST tag which is intensively used for large scale protein purification. Furthermore, the current construction also includes cell penetrating peptide poly-arginine-tag and secretion signaling peptide HlyA-tag which separated by Thrombin site for tag removal. Those tags not only increase the penetrating ability of apoptin, but also its purification. However, further comparison needs further analysis, such as western blotting and Bradford assay for quantitative analysis.

Growth inhibition effect of apoptin 8 Arg on the HeLa and widr cell lines
Following incubation with different concentration of Apoptin 8 Arg for 48 or 72 h, the proliferation of HeLa and Widr Cell were significantly inhibited in a concentration-dependent manner (see fig. 5 and fig. 6), the apoptin without a tag was used as a control. In this study, we conducted an experiment on in vitro cultured HeLa and Widr cells this experiment was also used to confirm apoptin activity for other cell lines such as EJ cells. Our results on HeLa and Widr cells confirmed similarly with Apoptin which tagged by TAT. Following treatment with Apoptin 8 Arg for 1 µg/ml had significantly effect on the HeLa cell and Widr cell [9]. Apoptin was confirmed induces tumor cell-specific cell death and a potential future anti-cancer therapeutic. Several strategies were developed, based on gene therapy or protein therapy. Gene therapy approach more developed compares with protein therapy that virus and cell is the common vectors [10,11]. Unfortunately, therapy that using protein are limited, it's because of the cost of expression and purification of protein are high compared with gene therapy.
Our study showed new strategy to expressed extracellular protein, by tagged the apoptin by HlyA the protein could exported to extracellular. To increase the protein expression in E. coli we optimized and synthesized apoptin gene. The apoptin was well expressed in E. coli, and have similar activity compared with original gene. This extracellular protein opens the continuous method to produce protein more cheap compare batch method.

CONCLUSION
Gene construction of modified apoptin has been done and confirmed by sequencing. Modified apoptin obtained and overexpressed on the BL21 CP cells. However, the apoptin is not obtained as an overexpressed protein in E. coli BL21 pLysS and DH5a cells pellet and a supernatant fraction. On the other hand, the possibility of Apoptin overexpression in media fraction still can be analyzed using a higher concentration of media precipitated.

ACKNOWLEDGMENT
This study was supported by an International collaboration grant from the state ministry of research and technology, Republik Indonesia.

CONFLICTS OF INTERESTS
Declared none