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

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

Heparins mediate the multimer assembly of secreted Noggin

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

Strategy for the purification of soluble fibronectin from human plasma, as ligand for affinity chromatography

El presente trabajo se desarrolló con el objetivo de purificar y caracterizar fibronectina soluble procedente de plasma para su posterior aplicación en la purificación de proteínas de adhesión. Se inmovilizó gelatina en Sefarosa 2BCL como soporte cromatográfico para la preparación de una columna. Se estudiaron diferentes alternativas de elución con arginina 0.5 M y 1M, con glicina -NaCl, acetato de sodio - NaCl y urea - NaCl, lo cual confirmó las ventajas en el uso de la arginina como agente eluyente. Se incorporó una segunda etapa de purificación en una columna de Heparina - Sefarosa 4B CNBr a partir de la que se obtuvo fibronectina con alto grado de pureza en estado nativo, comprobado por SDS-PAGE. La proteína purificada se inmovilizó en una matriz de Sefarosa con un grado de sustitución de 0,98 mg fibronectina/mL de gel lo cual permitió una capacidad de adsorción de gelatina libre con una recuperación de 8 mg (0,64 mg gelatina/mL de gel). La utilización de esta matriz permitirá su aplicación preliminar en la purificación de proteínas de adhesión que podrían ser usadas como antígenos en vacunas contra leptospirosis.Soluble fibronectin from human plasma was purified and characterized in order to be applied in the purification of adhesion protein. Gelatin was immobilized in a chromatographic bed as Sepharose 2B CL to prepare a column. Different alternatives of elution with 0.5 M and 1 M arginin, glicin- NaCl, sodium acetate - NaCl and urea - NaCl buffers, confirmed the advantages of the arginin as elution solution in this column. Second step of purification in a column of Heparin - Sepharose 4B CNBr was included to obtain the native fibronectin with high purity degree, verified by SDS-PAGE. Purified protein was immobilized in a matrix of Sepharose with a substitution grade of 0.98 mg fibronectin/mL of gel that permited an adsorption capacity of free gelatin to the column of 8 mg (0.64 mg gelatin/mL of gel). This matrix will allow its preliminary application in the purification of adhesion proteins which could be used as antigens in vaccines againt leptospirosis.Colegio de Farmacéuticos de la Provincia de Buenos Aire