The production of protein isolates from hexane-defatted ground yellow mustard meal

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The Swartz laboratory has developed a cell-free protein synthesis (CFPS) platform in which E. coli cell lysates are used for the rapid production of a variety of proteins including disulfide bonded proteins and virus-like particles (VLPs) as well as membrane proteins. This unique in vitro protein production platform is diluted approximately 20-fold compared to the E. coli cytoplasm, and translation rates during CFPS are about ten-fold slower than in living cells. The open cell-free system facilitates control of the protein synthesis environment (pH, redox, and component concentrations) and enables the supplementation of chaperones, disulfide bond isomerases, and other components in precise amounts during protein synthesis. These unique attributes enable the production of complex proteins, novel protein fusions and assemblies that are otherwise difficult, if not impossible to produce .
During the production of fusion proteins, the folding of one protein domain may interfere with the folding of its fusion partner. This problem can be worsened by the need for disulfide bond formation between cysteine residues in one or more of the fusion partners. It is therefore often desirable to produce proteins individually in active form, followed by coupling to produce bioconjugates. Post-translational conjugation technologies enable modification of proteins with a variety of molecules including small molecules, nucleic acids and peptides as well as other proteins. Protein-protein conjugation also facilitates the production of more complex proteinassemblies, and provides more control over the relative orientations of individual proteins in the resulting conjugates.
While several technologies have been developed for coupling or modification of proteins, they typically employ bi-functional linkers and a direct, one-step covalent coupling scheme is more desirable. Furthermore, the majority of linker-based methods involve N-hydroxysuccinimide or maleimide reactions which target amines or sulfhydryl groups, respectively. Since proteins typically have multiple amines and sulfhydryl groups, these methods offer less control over conjugation. Several non-natural amino acid (nnAA) incorporation schemes have been developed to facilitate site-specific conjugation or modification of proteins by means of a chemical reaction between unique azide and alkyne reactive groups. Azide-alkyne "click chemistry" is an attractive approach for chemical coupling because it is efficient, requires low reactant protein concentrations, and can be performed under physiological conditions. We adopted methods for the cell-free incorporation of azide and alkyne containing nnAAs in proteins to facilitate direct protein-protein conjugation using Cu(I) catalyzed "click" chemistry. The absence of the cell membrane barrier in CFPS enables convenient supplementation of the orthogonal components that are needed for nnAA incorporation.
Previous work in the Swartz laboratory has adapted the cell-free system for the site-specific incorporation of the tyrosine analogs p-azido-L-phenylalanine and p-propargyloxy-L-phenylalanine into proteins. This scheme, originally developed in E. coli by Dr. Peter Schultz at the Scripps Research Institute, enables the incorporation of a nnAA exclusively at any desired site in a protein. However, yields of protein containing tyrosine analogs are typically significantly lower than yields of the corresponding wild-type protein. We have now adopted the scheme for global replacement of methionine residues with L-azidohomoalanine and L-homopropargylglycine. Using this system, originally developed in E. coli by Dr. David Tirrell at the California Institute of Technology, multiple nnAAs can be introduced in place of methionine residues without significantly affecting protein production yields. The unique azide and alkyne reactive groups introduced in proteins using the above four amino acids facilitate direct protein-protein conjugation as well as site-specific post translational modification of proteins with nucleic acids and small molecules using azide-alkyne click chemistry.
This thesis describes efforts to simplify and streamline CFPS methods, to develop methods for direct protein-protein conjugation, and to utilize the unique capabilities of the CFPS platform for the fusion proteins production of and protein bioconjugates including: (1) Transducible transcription factors for the generation of induced pluripotent stem cells (iPSCs), (2) Tetanus toxin fragment C - tumor idiotype antibody fragment fusion protein vaccines for B cell lymphoma, (3) Gaussia luciferase—antibody fragment conjugates for the detection of tumor cells, and (4) Virus-like particle-based tumor idiotype vaccines for B cell lymphoma.
The design, cell-free production and functional evaluation of these novel fusion proteins and bioconjugates are described.