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.
Satakarni
Makkapati
M
School of Chemical Engineering and Analytical Science, The University of Manchester, PO Box 88, Manchester M601QD, United Kingdom.
Koutinas
Apostolis A
AA
Webb
Colin
C
Curtis
Robin
R
D019149:Bioreactors; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020689:Exopeptidases / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D025842:SUMO-1 Protein / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
D003470:Culture Media; C098329:EAK16 peptide; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D007544:Isopropyl Thiogalactoside; D020689:Exopeptidases; C400521:SUMO-1 C-terminal hydrolase; D005947:Glucose; D009584:Nitrogen