Indonesian Journal of Biotechnology VOLUME 24(1), 2019, 8–16 | RESEARCH ARTICLE Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production Achmad Rodiansyah 1,2* , Riyona Desvy Pratiwi 2 , Sabighoh Zanjabila 2 , and Asrul Muhamad Fuad 2 1 Department of Biology State University of Malang, Semarang Street No. 5, Malang 65145, Indonesia 2 Research Center for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Bogor KM.46, Cibinong 16911, Indonesia * Corresponding author: a.rodiansyah@yahoo.com SUBMITTED 14 December 2018 REVISED 16 April 2019 ACCEPTED 24 May 2019 ABSTRACT Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5α. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained. KEYWORDS Escherichia coli BL21(DE3); pET21b(+); recombinant human epidermal growth factor (rh-EGF); site-directed mutagenesis (SDM) 1. Introduction Human epidermal growth factor (h-EGF) is a small protein to stimulate cell proliferation, differentiation, viability in various cell types such as epithelial cells, fibroblast cells, endothelial cells including tumor cells with complex regu- latory mechanisms (Citri and Yarden 2006; Su et al. 2006; Xian 2007; Higashiyama et al. 2008; Zeng and Harris 2014). It is functional protein from derivate large precur- sor gene, this large precursor gene includes encoding mem- brane receptor, EGF-like repeats, and EGF or growth fac- tor family (Bell et al. 1986; Zeng and Harris 2014). This protein can be detected in saliva, milk, intestinal fluid, and others (Fisher and Lakshmanan 1990; Xian 2007; Dvorak 2010). h-EGF protein is composed of 53 amino acids with three disulfide bonds with conserved be cysteines from C1 to C6 specified in glycine and arginine. This mature pro- tein has molecule mass about 6.2 kDa (Savage et al. 1972, 1973; Zeng and Harris 2014). However, the rh-EGF used in this study has been added a methionine residue at the N-terminal for intracellular expression in E. coli. In our sequence, disulfide bonds were formed by binding of C7- C21; C14-C31, and C33-C43. In an in vivo study with pig and mice, it is showed that EGF can prevent and treat necrotizing enterocolitis and also be important in gastroin- testinal repair (Nair et al. 2008). EGF suppresses fibrosis in mice liver induced by thioacetamide (TAA) associated with inhibiting hepatic stellate cells (Huang et al. 2012). EGF is important in remodeling the cytoskeleton for en- docytosis and cell proliferation (Kharchenko et al. 2007). Growth factor family such as EGF contributed to wound healing in various tissues and their possibility play roles in therapy based on stem cells (Fu et al. 2002; Pikuła et al. 2015). h-EGF has been synthesized by several recombinant protein techniques due to its importance in treatments from h-EGF lack-related diseases. The recombinant h- EGF (rh-EGF) can be used for therapies in kidney destruc- tion diseases such as glomerular disease (Flamant et al. 2012; Klein et al. 2016). Moreover, rh-EGF for patients with diabetic ulcers with level 1–4 showed significant re- pair of injury (Putri and Sriwidodo 2016) and rh-EGF spray also significantly useful for reduced mucositis oral induced by radiotherapy (Wu et al. 2009). Indones J Biotechnol 24(1), 2019, 8–16 | DOI 10.22146/ijbiotech.41859 www.jurnal.ugm.ac.id/ijbiotech Copyright © 2019 THE AUTHOR(S). This article is distributed under a Creative Commons Attribution-ShareAlike 4.0 International license.