A promoter sequence for E. coli that has been demonstrated to function at a high level in the mammalian gut. If your project involves a probiotic or therapeutic protein that would benefit from being expressed in the digestive tract environment, this promoter will allow your protein to be continually expressed while your E. coli chassis is in that location.

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Figure 1: Diagram of RNA Polymerase About to Bind to Promoter Sequence for mRNA Synthesis

Note: Promoter sequence is for use in E. coli for constitutive expression of a gene of interest in the gut environment, such as for a probiotic product. Created with BioRender.com

The gene sequence for the human gastric intrinsic factor (GIF), a protein involved in vitamin B12 absorption, has had the codons optimized for expression in an E. coli chassis. Vitamin B12 cannot be directly absorbed into intestinal parietal cells and must first be bound to GIF for transport into the cell. As GIF is normally produced by human cells, the unaltered gene sequence contains codons that are infrequently used in E. coli, affecting the synthesis of the protein, which is why we optimized the gene sequence for codons commonly used by E. coli.

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Figure 2: Crystal Structure of Human Gastric Intrinsic Factor With Cobalamin Bound

Note: Ribbon diagram of the hGIF protein with a ball and stick diagram of the bound vitamin B12 (Cbl) molecule, based off X-ray crystallography data, forming an hGIF-Cbl complex (Mathews et al., 2007).

This signal peptide (PelB) and upstream translation initiation region (TIR) (contains Shine-Delgarno sequence) has been evolved to increase the expression of periplasmic proteins when bound to the N-terminal. The TIR includes the beginning of the signal peptide sequence as it was found that this part of the sequence was important for translation initiation, as well as the upstream untranslated region. Disulphide bonds do not generally form correctly in the cytosolic environment of E. coli, so a signal peptide was required, which is able to efficiently get the protein transported to the periplasm where environmental conditions are more favourable to proper protein folding for the gastric intrinsic factor (GIF) protein. Mirzadeh et al. (2020) were able to evolve this PelB signal peptide to increase the periplasmic export of human growth hormone in E. coli, which is a different human protein but one that also required proper disulphide bond formation to function correctly.

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Figure 3: Protein with N-terminal signal peptide

Note: N-terminal signal peptides, such as PelB, get cleaved from the rest of the polypeptide at the inner membrane. Created on BioRender.com

The creation of this part started with the identification of the amino acid sequence for the hGIF protein, which was accomplished by searching the National Center for Biotechnology Information (NCBI) database (NCBI, n.d.; Uniprot KB: P27352). After obtaining the amino acid sequence, IDT’s codon optimization tool was used to optimize the codons for expression in E. coli (IDT, n.d.). Codon optimization is important for efficient expression of recombinant proteins because of codon bias caused by tRNA abundance differences between species (Zalucki & Jennings, 2007). After the hGIF DNA sequence was optimized, it was important to ensure that it would get expressed and transported to the periplasm for proper protein folding. This was accomplished through a literature review which found an evolved PelB N-terminal signal peptide that demonstrated efficient expression and transport of the human growth factor (hGF) protein in an E. coli chassis (Mirzadeh et al., 2020). This was important as the hGIF protein contains disulphide bonds, like hGF, which do not form properly in the reducing environment of the E. coli cytosol generating improperly folded, non-functional proteins (Katzen & Beckwith, 2002). The cytosol of E. coli contains multiple thio-reducing enzymes, which is why E. coli exports its own native proteins with disulphide bonds to the periplasm (Katzen & Beckwith, 2002).

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Figure 4: hGIF Translation and Transportation to the Periplasm due to PelB Signal Peptide

Note: Schematic of N-terminal PelB signal peptide recruiting a shuttle protein that recognizes its amino acid sequence and shuttles the peptide to the inner membrane where a transporter cleaves off the PelB peptide and transports the remainder of the protein (hGIF) to the periplasm where it can fold into its functional conformation. Created with BioRender.com

This is the full protein coding sequence, translation initiation region (TIR), and signal peptide sequence (PelB) for human gastric intrinsic factor (GIF) expression in E. coli while in the human gut. The gene sequence has been codon optimized for expression in E. coli with an evolved signal peptide on the N-terminal to optimize the export of the protein to the periplasm for proper folding. A TIR sequence is upstream of the gene and signal sequence (the start of the signal peptide gene sequence is important for efficient translation and is part of the TIR). Restriction sites can be added to both ends of the gene sequence to facilitate cloning, we used Bsa-I HFv2 restriction sites for Golden Gate assembly with NEB's Golden Gate kit, which included an appropriate vector, pGGAselect.