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1.
Nat Protoc ; 19(1): 127-158, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37974029

ABSTRACT

The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENPEuB. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.


Subject(s)
Peptides , Proteins , Humans , Proteolysis , Recombinant Fusion Proteins , Chromatography, Affinity/methods
2.
Matrix Biol Plus ; 14: 100111, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35619988

ABSTRACT

In pancreatic ductal adenocarcinoma (PDAC), the abundant stromal cells which comprise the tumor microenvironment constitute more than 90% of the primary tumor bulk. Moreover, this desmoplastic environment has been found to be three times stiffer than normal pancreas tissue. Despite the importance of studying the desmoplastic environment of PDAC, there is still a lack of models designed to adequately recapitulate this complex stiff microenvironment, a critical hallmark of the disease that has been shown to induce chemoresistance. Here, we present a bio-mimetic, 3-dimensional co-culture system that integrates tumor organoids and host-matching stromal cancer associated-fibroblasts (CAFs) that recapitulates the complex, fibrotic matrix of PDAC using advanced biomaterials. With this model, we show that matrix-activated CAFs are able to "re-engineer" the fibrotic environment into a significantly stiffer environment through lysyl-oxidase dependent crosslinking. Moreover, we show that culture of CAFs in this model leads to an increase of exosomes; extracellular vesicles known to promote chemoresistance. Finally, using previously identified exosome inhibitors, climbazole and imipramine, we demonstrate how abrogation of exosome hypersecretion can reduce matrix stiffness-induced chemoresistance. These data highlight the importance of the development of new models that recapitulate not only the cellular composition found in PDAC tumors, but also the biophysical stresses, like stiffness, that the cells are exposed to in order to identify therapies that can overcome this critical feature which can contribute to the chemoresistance observed in patients. We believe that the 3D bio-mimetic model we have developed will be a valuable tool for the development, testing, and optimization of "mechano-medicines" designed to target the biophysical forces that lead to tumor growth and chemoresistance.

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