Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), is a multifaceted growth factor with diverse biological functions. KGF is secreted exclusively by mesenchymal cells and plays a pivotal role in embryonic development, cell proliferation, and differentiation (Jang et al., 1997). It is essential for branching morphogenesis and keratinocyte development (Ornitz et al., 1996, Zhang et al., 2006) exerts substantial paracrine effects on normal epithelial cell proliferation (Alpdogan et al., 2006, Hille et al., 2010). KGF is a member of the expansive FGF family, which comprises 22 known members (Beenken and Mohammadi, 2009, Ornitz and Itoh, 2001, Yun et al., 2010). It specifically interacts with the b isoforms of FGFR1 and FGFR2 (FGFR1b/FGFR2b) found on epithelial cells (Zinkle and Mohammadi, 2019). Structurally, KGF consists of 163 amino acid residues, two intramolecular disulfide bonds, and two glycosylation sites (Hsu et al., 1998). Post-translational truncation at Arg23 can produce a 140-residue, non-glycosylated form of KGF with a single disulfide bond; notably, both truncated and non-truncated forms of KGF are biologically active (Hsu et al., 1998). Furthermore, deletions at the N-terminus have been shown to significantly impact receptor binding and biological activity (Hsu et al., 2006, Nybo et al., 1997, Ron et al., 1993). However, despite its importance, the three-dimensional structure of human KGF has not been determined to date.

Recombinant truncated KGF, known as Palifermin, has received FDA approval for its efficacy in reducing both the severity and duration of oral mucositis following intensive chemotherapy and radiotherapy treatments (Spielberger et al., 2004). Furthermore, recombinant KGF demonstrates promising applications in the differentiation of human pluripotent stem cells into pancreatic progenitors and islet-like organoids (Yoshihara et al., 2020), as well as in wound healing and hepatocyte protection (Feng et al., 2014, Xue et al., 2014). However, the efficient production of recombinant KGF remains a challenge. Previous attempts to produce KGF in Escherichia coli have resulted in low expression levels and protein aggregation (Bare et al., 1994, Ron et al., 1993). Various strategies, including the use of different fusion tags and culture media modifications, have yielded only modest improvements (Kim et al., 2023a, Kim et al., 2023b, Luo et al., 2004, Wu et al., 2009). On the other hand, eukaryotic expression systems, although more effective, are costly and time-consuming (Bahadori et al., 2018, Dudognon et al., 2014, Feng et al., 2014, Xue et al., 2014).

The present study explored the use of two protein tags, the lateral half of protein disulfide isomerase (PDI), called PDIb'a'—and maltose binding protein (MBP) known for enhancing recombinant protein expression and solubility in E. coli (Lee et al., 2021, Nguyen et al., 2021)for enhancing the production and solubility of recombinant KGF in different strains of E. coli. We genetically fused PDIb'a', MBP, and His (H) tags to the N-terminus of KGF to create H-PDIb'a'-KGF, H-MBP-KGF, and H-KGF constructs, respectively. These fusion proteins were expressed in multiple E. coli strains to assess their solubility and expression levels. Subsequently, we purified H-PDIb'a'-KGF and removed its tag to assess its biological activity.