Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

Aniel Moya-Torres,Fabian Heide,Markus Meier,Thomas Imhof,Denise Nikodemus,Jörg Stetefeld,Scott Legare,Manuel Koch,Monika Gupta,Natalie Krahn

doi:10.1007/s00253-021-11438-0

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.Key points• Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1• Biolayer interferometry allows target protein quantitation in expression media• High production yields in the bioreactor do not impair Netrin-1 proteoglycan qualityGraphical abstract

Highlights

Studying the structures and functional behavior of extracellular matrix (ECM) proteins requires large quantities of proteins.studying biophysical behavior and determining protein structures can require milligram amounts if a system is to be fully characterized
The Netrin family of proteins has been studied for some time, the guidance mechanisms are still not fully understood which would allow for the design of effective treatment for various physiological disorders (Arakawa 2004; Méneret et al 2017)
The transient expression of the full-length (FL) Netrin-1 was relatively low and purification was difficult to achieve due to instability of the protein in solution

Summary

Introduction

Studying biophysical behavior and determining protein structures can require milligram amounts if a system is to be fully characterized One such example is the ECM proteoglycan Netrin-1; Netrin-1 is an axon guidance cue, well. Appl Microbiol Biotechnol (2021) 105:6047–6057 known for its various signaling functions in cell proliferation, migration, and survival through transmembrane receptors (Boyer and Gupton 2018). These protein-protein interactions can initiate intracellular signaling cascades causing attraction, repulsion, and/or apoptosis (Arakawa 2004) which has significant applications in physiological diseases such as cancer (Mehlen and Furne 2005). Production of Netrin-1 poses some difficulties as glycosylation patterns vary among collections which affects the folding, solubility, and potential binding to receptors

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Conclusion