Universität zu Köln

Moya-Torres, Aniel ORCID: 0000-0001-7935-2591, Gupta, Monika, Heide, Fabian ORCID: 0000-0002-6849-3177, Krahn, Natalie, Legare, Scott, Nikodemus, Denise, Imhof, Thomas, Meier, Markus, Koch, Manuel ORCID: 0000-0002-2962-7814 and Stetefeld, Jorg (2021). Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor. Appl. Microbiol. Biotechnol., 105 (14-15). S. 6047 - 6058. NEW YORK: SPRINGER. ISSN 1432-0614

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Abstract

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Moya-Torres, Aniel UNSPECIFIED orcid.org/0000-0001-7935-2591 UNSPECIFIED Gupta, Monika UNSPECIFIED UNSPECIFIED UNSPECIFIED Heide, Fabian UNSPECIFIED orcid.org/0000-0002-6849-3177 UNSPECIFIED Krahn, Natalie UNSPECIFIED UNSPECIFIED UNSPECIFIED Legare, Scott UNSPECIFIED UNSPECIFIED UNSPECIFIED Nikodemus, Denise UNSPECIFIED UNSPECIFIED UNSPECIFIED Imhof, Thomas UNSPECIFIED UNSPECIFIED UNSPECIFIED Meier, Markus UNSPECIFIED UNSPECIFIED UNSPECIFIED Koch, Manuel UNSPECIFIED orcid.org/0000-0002-2962-7814 UNSPECIFIED Stetefeld, Jorg UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-586916
DOI:	10.1007/s00253-021-11438-0
Journal or Publication Title:	Appl. Microbiol. Biotechnol.
Volume:	105
Number:	14-15
Page Range:	S. 6047 - 6058
Date:	2021
Publisher:	SPRINGER
Place of Publication:	NEW YORK
ISSN:	1432-0614
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language GENE-EXPRESSION; CELL-CULTURE; MUTATIONS; RECEPTORS; STRESS; GROWTH Multiple languages Biotechnology & Applied Microbiology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/58691