No 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 quality Graphical abstract

Extracellular matrix proteins are most often defined by their direct function that involves receptor binding and subsequent downstream signaling. However, these proteins often contain structural binding regions that allow for the proper localization in the extracellular space which guides its correct function in a local and temporal manner. The regions that serve a structural function, although often associated with disease, tend to have a limited understanding. An example of this is the extracellular matrix protein Noggin; as part of the bone morphogenetic protein inhibitor family, Noggin serves a crucial regulatory function in mammalian developmental stages and later periods of life. Noggin's regular function, after its expression and extracellular release, is mediated by its retention in close proximity to the cellular surface by glycosaminoglycans, specifically heparin and heparan sulfate. Using a biophysical hybrid method approach, we present a close examination of the Noggin heparin binding interface, study its dynamic binding behaviors and observe supramolecular Noggin assemblies mediated by heparin ligands. This confirms previously suggested models of non-covalent protein assemblies mediated through glycosaminoglycans that exist in the extracellular matrix. Further, structural analyses through molecular dynamics simulations allowed us to determine contribution energies for each protein residue involved in ligand binding and correlate this to disease associated mutation data. Our combination of various biophysical and computational methods that characterize the heparin binding interface on Noggin and its protein dynamics expands on the functional understanding of Noggin and can readily be applied to other protein systems.