|Title:||Development of a refolding and capture protocol for HRP IBs produced in E. coli||Language:||English||Authors:||Ebner, Julian||Qualification level:||Diploma||Keywords:||Protein Refolding; Horseradish peroxidase; Inclusion body processing; Capture and Concentration; Fed-Batch refolding||Advisor:||Spadiut, Oliver||Assisting Advisor:||Humer, Diana Claudia||Issue Date:||2021||Number of Pages:||52||Qualification level:||Diploma||Abstract:||
While Horseradish Peroxidase (HRP) is an already established enzyme used in diagnostic kits and immunoassays, it saw a rising interest in the past years due to the possible application in targeted cancer treatment. This requires a reliable supply of well-defined HRP. However, it is currently only produced from its natural source, the horseradish root, where it occurs in at least 15 different isoforms and exhibits an inhomogeneous plant glycosylation pattern. While several of studies have reported the recombinant production in a variety of hosts, none of them have presented a viable alternative to current production, mainly due to low yields. One organism that can potentially produce HRP up to high titers is E. coli. In this case, though, HRP is not produced in its active form, but as Inclusion bodies (IBs). Refolding yields of HRP IBs are low, mitigating the high titers achieved during the fermentation.In this work, the unit operations solubilization, refolding and capture were investigated in order to obtain active HRP from HRP IBs. Three distinct sections are presented here, they are, however, not equivalent to the unit operations. Instead, for the first section, the unit operations solubilization and refolding were investigated in an integrated approach using a small-scale Design of Experiment approach. The main focus was put on the redox conditions during solubilization and refolding, which enables the formation of disulfide bonds and proved to be essential for the correct folding of HRP. For the second section, a scale-up to a refolding reactor was performed. This allowed for a controlled environment and monitoring of the redox level during refolding. In addition, a fed batch refolding approach was tested in order to enhance refolding yields. With the tried methods, however, no improvement of the refolding yield could be achieved in comparison to the batch refolding mode. For the third section, a suitable capture step using salt precipitation of impurities followed by a hydrophobic interaction chromatography was investigated.Based on the results of these three parts, a process which resulted in a specific activity of 980 U/mg and a purity of 98% according to SEC-HPLC could be established. Furthermore, a yield of 567 mg active HRP / L fermentation broth could be achieved. Additionally, the advantages of an integrated approach, especially for solubilization and refolding, could be shown in this work.
|DOI:||10.34726/hss.2021.87273||Library ID:||AC16131954||Organisation:||E166 - Institut für Verfahrenstechnik, Umwelttechnik und technische Biowissenschaften||Publication Type:||Thesis
|Appears in Collections:||Thesis|
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checked on Feb 27, 2021
checked on Feb 27, 2021
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