Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris.
Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic.
Kristína
K
Melicherová
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic. krahulec@fns.uniba.sk.
Ján
http://orcid.org/0000-0002-9719-3675
J
Krahulec
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic.
Martin
M
Šafránek
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic.
Veronika
V
Lišková
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic.
Diana
D
Hopková
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic.
Diana
D
Széliová
Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic.
Ján
J
Turňa
D002384:Catalysis; D004765:Enteropeptidase / Q000096:biosynthesis* / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D004765:Enteropeptidase
Activity; Enterokinase; Pichia pastoris; Specific growth rate