N-region of Cry1Ia: a novel fusion tag for Escherichia coli and Pichia pastoris

When expressing a recombinant protein in a prokaryotic system, specific peptides called fusion tags are often added to the N- or C-terminus of the protein. Fusion tags have numerous functions, with some increasing the expression of recombinant proteins, others increasing their solubility, and still others that can be used for subsequent protein purification (Costa et al., 2014, Ki and Pack, 2020, Vandemoortele et al., 2019). Secretory signal peptides (SPs) or secretory proteins can also be used as fusion tags to translocate target proteins into the periplasmic space of gram-negative bacteria or to the space outside cells (Gupta and Shukla, 2016, Kleiner-Grote et al., 2018, Overton, 2014). Transmembrane translocation offers several important advantages over intracellular expression strategies. First, it prevents cytotoxicity caused by excessive intracellular accumulation of recombinant proteins or unintended degradation by intracellular proteases, thereby increasing yield (Freudl, 2018, Ignatova et al., 2003, Mergulhão et al., 2005). Second, recombinant protein secretion prevents the formation of inclusion bodies in the cytosol (Jeong and Lee, 2001, Loo et al., 2002). Third, the folding of some recombinant proteins requires disulfide bonds, but the reducing environment of the cytoplasm is not conducive to the formation of disulfide bonds (Manta et al., 2019). Therefore, transferring recombinant proteins to the periplasmic space or extracellular environment can facilitate the correct folding of these proteins (Berkmen, 2012, Ke and Berkmen, 2014, Shokri et al., 2003).

In prokaryotic systems, specific SP do not behave identically for all recombinant proteins (Esposito and Chatterjee, 2006). Under the same conditions, the SP Iasp kept approximately half of the IeGFP protein soluble, whereas both I-GDF8 and I-MMP13 formed inclusion bodies (Gao et al., 2020). Mirzadeh et al. (Mirzadeh et al., 2020) fused β-lactamase with SPs of maltose-binding protein (MBP), outer membrane protein A (OmpA), alkaline phosphatase (PhoA), disulfide oxidoreductase (DsbA), and pectate lyase B (PelB), respectively, and expressed the fusion proteins in Escherichia coli. They found that the former four SPs resulted in high expression levels of β-lactamase, whereas PelB did not, and only MBP and PelB could direct β-lactamase secretion to the periplasm. The lengths and amino acid sequences of SPs vary widely; however, most possess three canonical regions, namely the N-, H-, and C-regions (von Heijne, 1990). The N-region is more hydrophilic and has a positive net charge, the H-region is more hydrophobic, and the C-region is less hydrophobic and contains the signal peptidase recognition sequence. The hydrophobic region of SPs can interact with the hydrophobic region of cargo proteins, thereby affecting the correct folding of the proteins and leading to a decrease in their thermodynamic stability (Park et al., 1988). The SPs of MBP or pelB significantly interfered with the thermodynamic stability of MBP and Trx when retained in the final products (Beena et al., 2004, Krishnan et al., 2009, Singh et al., 2013). This feature may also be the basis for the use of SPs as inclusion body tags. For example, the SP of trimethylamine N-oxide reductase (torA) of E. coli and its tandem polypeptides can convert various proteins, including thioredoxin A (TrxA) and MBP, into inclusion bodies when placed at either the N-terminal or C-terminal end (Jong et al., 2017).

SPs in prokaryotic and eukaryotic cells share similar properties, but the protein transport pathways differ. The presence of multiple membrane-bound organelles and post-translational modification mechanisms in eukaryotic cells complicates the protein sorting system. Nevertheless, secretory signals of prokaryotic and eukaryotic cells are believed to be distinguishable from each other, especially when eukaryotic proteins are recombinantly expressed in bacteria (Humphreys et al., 2000, Malik et al., 2007). However, the translocation efficiency of different secreted proteins considerably varies. In eukaryotic cells, several secretory proteins from bacteria are either less efficiently or not at all recognized by the translocation pathway. For example, E. coli β-lactamases can be translocated into Canis lupus familiaris pancreatic microsomes in vitro (Müller et al., 1982), and Xenopus laevis oocytes recognized their SP (Wiedmann et al., 1984), but in Saccharomyces cerevisiae, only 10% of the product was processed (Roggenkamp et al., 1983, Roggenkamp et al., 1981). The α-amylase from the gram-positive bacterium Bacillus licheniformis can efficiently translocate across the membrane in Pichia pastoris (Paifer et al., 1994). There is evidence that plants recognize the SPs of prokaryotic systems. In Zea mays and Arabidopsis thaliana, the E. coli heat-labile enterotoxin B subunit (LT-B) SP (BSP) can direct green fluorescent protein (GFP) into the endoplasmic reticulum (Moeller et al., 2009). Notably, LT-B protein with complete BSP sequences also accumulates in the starch grains of maize seeds through an unknown mechanism (Chikwamba et al., 2003, Moeller et al., 2009). Thus, the potential of using SPs from prokaryotic sources as fusion tags in eukaryotic cells is also promising.

In our previous study, we showed that in E. coli, the SPs of the Bacillus thuringiensis (Bt) insecticidal protein Vip3A (Vasp) caused nearly all of the more soluble enhanced GFP (eGFP) to accumulate as inclusion bodies, but its expression level was significantly higher (Gao et al., 2022). The SPs of another Bt insecticidal protein Cry1Ia (Iasp) not only increased the expression level of eGFP but also showed less negative effects on its solubility (Gao et al., 2020). Both SPs promoted the expression of various exogenous proteins. In addition to using the entire SP sequence, modification of the natural SPs can also significantly improve the secretory expression of the protein (Low et al., 2013). Previously, we deleted the hydrophobic region of Vasp and this truncated SP (VN) could still maintain a high expression of recombinant protein while also reducing the negative effects on solubility (Gao et al., 2022). In this study, we compared the effects of Vasp, Iasp, and their N-region sequences on recombinant protein expression. In addition, we investigated the expression of Vasp- or Iasp-directed eGFP in P. pastoris GS115 to highlight a novel case for the mutual recognition of SPs between prokaryotes and eukaryotes. This study shows the potential application of the hydrophilic N-region of proteins as a fusion tag.

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