Kactus proprietary platform, SAMS™ (Structure Aided Multiplex Screening), enables high success rates for challenging proteins by combining protein structural design with robust protein expression screening.
Structure based-rational design is the essential part of protein engineering, as protein secondary and tertiary structures have direct impacts on protein expression, folding and function. In addition to common considerations like secretive signal peptide, fusion tag and codon optimization, scientists in Kactus Biosystems perform comprehensive structural informatics, including 3D modeling and bioinformatics, into protein designs.
For protein expression, there is no one-size-fits-all expression condition. Therefore, screening approaches are quite necessary to determine optimal condition ranges for the target proteins. Scientists in Kactus Biosystems creatively designed high throughput protein expression screening system, coupled with proprietary transfection reagents and high cell density cell line Expi293, making Kactus’s screening system powerful – in fact unique to the industry.
● MembraneXTM Engineered chimeric antigen design and production;
● UltraActiveTM Multimeric protein- better cell based bioactivity;
● Proprietory MHC I/MHC II design and tetramer assembly ;
● ImmunoProTM Antigen display on nanoparticle as immunogen;
● Challenging protein expression and screening
Based on protein structural constraints along with structural modeling against the membrane bound CD20, Kactus Biosystems successfully produced CD20 recombinant proteins harboring the functional epitopes of CD20. The EC50 of CD20 is similar to the reported EC50 value for rituximab, hence suitable for drug discovery, screening and quality control.
Structure based rational design is widely applied in generating membrane protein mimic antigen and epitope scaffolding protein. By introducing proper structure constraints, the antigen or epitopes can form correct conformations, hence be functinal relavant.
In literature, E. coli expression and in vitro protein refolding is a common method to produce MHC-I complex. Using structure-guided MHC-I design approach coupled with multiplex screening, Kactus succeeded in expressing biotinylated MHC-I complex in mammalian cell Expi293 on a single step. The resulting MHC-I complexes show high affinities with benchmark antibodies and have low endotoxin levels – ideal for endotoxin sensitive cell-based assay.
Biotinylated MHC-I can bound to streptavidin and form MHC tetramer. Kactus developed in house streptavidin / fluorophore-labeled streptavidin as well as optimized method for tetramer formation. Kactus has succeeded in providing HLA-A, HLA-G, HLA-E MHC-I tetramers to customers. Using similar strategies, Kactus is capable of producing MHC-II tetramers.
Virus-Like Particles (VLPs; aka nanoparticles) are highly organized spheres that self-assemble. Kactus scientists have produced a panel of nanoparticles ideal for displaying various antigens, including transmembrane proteins. Because VLPs elicit innate and cognate immune responses in hosts, the structure-guided VLP display platform is ideal for the antibody discovery campaign (i.e., immunization).
Considering post translational modification of target antigen and nanoparticle yield, both E.coli and mammalian Expi293 expressions are commonly employed in Kactus.
To increase the production of DLL3, we have executed two rounds of Multiplex Screening with the DLL3 expression plasmid. As a result, the DLL3 expression level increased 10 folds compared to typical/initial expression condition. Kactus Biosystems uses its Multiplex Screening for all protein constructs, by default.
More successful cases incude PSMA, GDF15, Integrin proteins et.al. Kactus high through-put screening system is critical in achieving cost effective production of challenging proteins.
For bispecific antibody or multi-subunits protein expression, 2 or 3 plasmids cotransfection is required. Optimal plasmid ratio can be empirically determined in Kactus screening system. Expression screening results in turn provide valuble information on improving construct design.
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