Recombinant Production of Peptide C-Terminal α-Amides Using an Engineered Intein

Abstract

Peptides are of increasing interest as therapeutics in a wide range of diseases, including metabolic diseases such as diabetes and obesity. In the latter, peptide hormones such as peptide YY (PYY) and pancreatic peptide (PP) are important templates for drug design. Characteristic for these peptides is that they contain a C-terminal that is α-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a larger scale. Initially, we experienced challenges with hydrolysis of the intein fusion protein, which was reduced by a T3C mutation in the intein. Subsequently, we further engineered the intein to decrease the absolute size and improve the relative yield of the PYY derivative, which was achieved by substituting 54 residues of the 198 amino acid intein with an eight amino acid linker. The optimized intein construct was used to produce the PYY derivative under high cell density cultivation conditions, generating the peptide thioester precursor in good yields and subsequent amidation provided the target peptide.

An Evolved Mxe GyrA Intein for Enhanced Production of Fusion Proteins

“…The evolved 202–08 intein did not appear to affect cleavage efficiency compared with the wild-type intein, and these cleavage efficiencies are consistent with those observed for bacterially produced proteins. 7 , 16 , 53 Furthermore, the release could possibly be enhanced by optimizing the carboxy-terminal residue of the target protein, which has previously been shown to impact the cleavage efficiency. 53 , 54 Regardless, the small amount of uncleaved material is not chemically functionalized and would not impact many downstream applications like antibody immobilization, but the uncleaved material could be removed by depletion via histidine tag purification if desired.…”

“…This smaller intein variant led to a 1.2-fold increase in intein-peptide fusion production in E. coli . 16 …”

“… 7 , 9 − 15 One disadvantage of cytoplasmic expression is the formation of insoluble inclusion bodies that contain inactive protein-intein fusions, therefore requiring solubilization of the inclusion bodies and refolding of the protein. 7 , 9 , 10 , 14 − 16 Glutathione redox buffers that are typically used to refold disulfide-containing proteins such as antibodies can react with the thioester intermediate formed by the intein, thereby releasing it from the target protein and forming an unstable glutathione thioester on the carboxy-terminus of the target protein. 10 This unstable glutathione thioester can subsequently be hydrolyzed leading to loss of the thioester functionality.…”

Impacts of the −1 Amino Acid on Yeast Production of Protein‐Intein Fusions

“…Interestingly, no autocleavage was observed when the −1 residue was mutated to Phe and more limited autocleavage was observed for Gln. It has previously been reported that engineering inteins for a particular property can lead to unexpected changes in cleavage efficiency and behavior, therefore it is possible that the engineering of 202‐08 for increased secretion led to a more promiscuous cleavage behavior. Although autocleavage was limited when using the wild‐type intein compared with the 202‐08 intein, total secretion levels of the protein‐intein fusion were considerably lower for the wild‐type intein, similar to values reported previously .…”

“…13,14 The secreted product can then be assayed with an anti-FLAG tag Western blot to identify the fraction of produced protein that has undergone successful cleavage. As intein cleavage efficiency and specificity are often difficult to predict, [26][27][28] and mutations to the Mxe GyrA intein can potentially affect intein cleavage activity, 26,29,30 the effects of −1 residue substitution were also screened for the presence of undesirable cleavage events that occur in the absence of MESNA (termed autocleavage). These could occur within the yeast secretory pathway or in the culture medium.…”