SPI “sandwich”: Combined SUMO‐Peptide‐Intein expression system and isolation procedure for improved stability and yield of peptides

Abstract

Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin-like modifier protein (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a "sandwiched" fusion protein of the form: His 6 -SUMO-peptideintein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17-fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation.

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides

“…That the entire system (containing Bsa BI blunt restriction enzyme site) can be transferred to any vector, we confirmed by the construction of additional pQE_Ek expression vector (Figure 1). Based on the latest results in the development of new improved expression vectors, we can conclude that the improvement of expression vectors will most likely go in the direction of reducing the toxicity of expressed AMPs (sandwich tag vector construction) and those contributing to AMP modification to increase stability and functionality (Lamer et al, 2022; Zhu et al, 2021). The improvements we have presented in this study can be combined with other improvements (most are compatible) in the construction of new expression vectors with multiple tags (sandwich tag) and with enzymatic modifications and processing.…”

Methods for Recombinant Production and Purification of Peptides as SUMO‐Peptide‐Intein Fusion Proteins to Protect from Degradation

“…MALDI‐TOF MS of SUMO‐peptide fusion protein and cleaved intein after overnight incubation of an SPI fusion protein in 100 mM DTT. Example mass spectrum for purification of the bacteriocin peptide faerocin MK (Lamer et al., 2022). Figure adapted from Lamer et al.…”

“…kDa = Molecular weight standards in kilodaltons. Example results for purification of the bacteriocin peptide leucocin A (Lamer et al., 2022). Lanes: MW, PageRuler Prestained Protein Ladder; 1, SUMO‐peptide fusion protein before addition of SUMO protease; 2, after incubation of SUMO‐peptide fusion protein with SUMO protease for 2 hr; 3, flowthrough from the final Ni‐NTA column; 4, wash (20 mM imidazole) final Ni‐NTA column; 5, elution (500 mM imidazole) of final Ni‐NTA column.…”

“…Figures were created with Biorender.com. Figures 3, 4, 5, 6, and 8 were adapted from Lamer et al (2022) with permission.…”

“…The small ubiquitin‐like modifier (SUMO) is a common fusion tag choice for peptides, as cleavage is completed by addition of a highly specific SUMO protease that leaves no extra residues behind on the peptide of interest (Butt, Edavettal, Hall, & Mattern, 2005). However, isolation of peptides as SUMO fusion proteins can be complicated by degradation of the fusion protein during expression in E. coli (Lamer et al., 2022). This can be a major problem if a large amount of peptide is needed for downstream applications, as the final yield of purified peptide can be quite low.…”