Enrichment of fermentation media and optimization of expression conditions for the production of EAK₁₆ peptide as fusions with SUMO

Abstract

EAK(16) (AEAEAKAKAEAKAEAK) belongs to a novel class of self-assembling peptides, which is being investigated in research and industry. SUMO belongs to the ubiquitin class of proteins and is a promising fusion partner currently in use. In this study, EAK(16) peptide fusions with hexa-histidine tagged SUMO have been constructed using Escherichia coli based pET expression vector. Intracellular expression of the SUMO-EAK(16) fusion using LB media has been optimized. Low-cost complex media (fungal autolysates, wheat and gluten hydrolysates) produced via a novel wheat-based biorefinery have been used as alternative fermentation media to LB. Shake flask cultures using either enriched LB or complex wheat-derived media containing 2 g/L of glucose resulted in intracellular SUMO-EAK(16) fusion protein production of approximately 250 mg/L fermentation volume which corresponded to 30-35% of the total bacterial protein expressed being the fusion protein. Fusion protein productivities up to five times higher were achieved when using a bioreactor.

Recombinant production of self-assembling β-structured peptides using SUMO as a fusion partner

“…SUMO-peptide fusions were recovered at 0.37 to 0.5 g/L with HPLC purification from the SUMO protease cleavage reaction resulting in yields of soluble P 11 -peptides of between 18 and 35 mg/L demonstrating that we have achieved good levels of recovery of purified peptides. It is likely this can be increased significantly as the maximum yield of SUMO protein achieved was 1.5 g/L compared to 637 mg/mL reported by Li et al, [28] using a similar expression strategy, while for EAK16 peptide production 250 mg/L of fusion protein was reported by Satakarni et al, [22] by IPTG induction.…”

“…The tertiary structure of SUMO, rather than a sequence motif, is recognised and cleaved by SUMO protease which cleaves after two Gly residues at the C-terminus of SUMO thus releasing the associated protein or peptide with a native N-terminus. SUMO has been successfully used for production of vesicle forming peptides [21] and another self-assembling peptide EAK 16 [22]. …”

Microbial biodegradable plastic production from a wheat-based biorefining strategy

“…Starch-and gluten-rich streams are used for the production of hydrolysates that are subsequently used for the production of PHB and recombinant proteins. Shake flask cultures of recombinant E. coli carried out in both enriched LB and gluten hydrolysates containing 2 g/l of glucose resulted in the production of similar intracellular SUMO-EAK 16 fusion protein (30-35% of the total bacterial protein) [37]. Future research should focus on the identification and optimisation of recombinant protein production from gluten hydrolysates.…”