Biology subjects

Wall, V.

Publications and source records attributed to Wall, V..

3 recordsLinked to original sources

bioRxiv2022

Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinantleucine-rich repeat proteins and protein complexes

The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins. HighlightsO_LIImproved yields and purity of LRR proteins and multiprotein complexes through chaperone co-expression, DNA construct design, and use of polycistronic baculovirus. C_LIO_LIOver 300-fold yield increase of SMP protein complex from 0.1 mg/L to 32.6 mg/L. C_LIO_LISUGT1 co-expression is a highly effective technique for recombinant LRR protein expression and purification. C_LIO_LIPolycistronic baculovirus infection is ideal for production of multiprotein complexes C_LI

biochemistry↗

bioRxiv2020

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: O_LIImproved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters C_LIO_LITwo versions of RBD show different sensitivity in serology assays C_LIO_LIYields of greater than 50 mg/l obtained under optimal conditions C_LIO_LIMagnetic bead purification technology improves throughput of protein production C_LI

biochemistry↗

bioRxiv2020

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots. HighlightsO_LIImproved yields of SARS-CoV-2 spike protein through modification of expression and purification parameters C_LIO_LIYields of greater than 5 mg/l obtained for VRC spike under optimal conditions C_LIO_LISpike protein quality was validated by QC methods to ensure utility in serology assays C_LI

biochemistry↗