Production of recombinant peptides as fusions with SUMO

Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.     

Enrichment of fermentation media and optimization of expression conditions for the production of EAK(16) peptide as fusions with SUMO

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.