University of Oulu

Incorporation of non-canoncical amino acids into recombinant human proteins heterologously expressed in E. coli by bioprocess parturbations

Saved in:
Author: Ongey, Elvis
Organizations: 1University of Oulu, Faculty of Biochemistry and Molecular Medicine, Biochemistry
Format: ebook
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 4 MB)
Persistent link: http://urn.fi/URN:NBN:fi:oulu-201406121759
Language: English
Published: Oulu : E. Ongey, 2014
Publish Date: 2014-06-12
Physical Description: 97 p.
Thesis type: Master's thesis
Tutor: Neubauer, Peter
Reviewer: Haikarainen, Teemu
Vasala, Antti
Description:
The purity of heterologous recombinant proteins is of utmost importance to the pharmaceutical sector since most are consumed as therapeutic agents by humans. Variability caused by co- and posttranslational modifications is a major concern in pharmaceutical production. In order to develop strategies which guarantee a homogeneous product in a robust production process, it is important to better understand the metabolic basis of the synthesis of related non-canonical amino acids. So far, studies have identified high glucose fluxes in connection to oxygen limitation and overexpression of leucine-rich proteins as possible reasons for the production of non-canonical amino acids and their incorporation into heterologous proteins expressed in Escherichia coli. The results presented in this work provide evidence that oscillations in the concentrations of glucose and oxygen as they occur in inhomogeneous industrial scale bioreactors potentiate the synthesis and incorporation of norvaline into the leucine-rich protein IL-2, heterologously expressed in E. coli W3110M, as observed in well-mixed homogenous cultures and perturbed shake flask cultivations. In order to represent the heterogeneities existing in large-scale bioreactors, two experimental setups were applied, using a simple shake flask scale-down model developed to monitor dissolved oxygen and pH online during a batch and fed-batch cultivation phases. Results here show that by applying repeated glucose pulses to the glucose limited culture, which consequently induce oscillations in dissolved oxygen, norvaline is accumulated. Analysis of inclusion bodies that resulted from the expressed IL-2 revealed the presence of norvaline in the protein. A higher concentration of norvaline was observed in the oscillating scale-down model compared to the non-perturbed culture, which suggests that the conditions as they typically occur in large scale bioreactors may be critical for product quality. The results and tools, developed in this work are a solid basis for future cell engineering approaches to overcome the challenges in view of product quality.
see all

Subjects:
Copyright information: This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.