Abstract: Compositions and methods for a nucleic acid amplification and diagnostic methods based therein, are disclosed. The compositions include purified Thermus aquaticus DNA polymerase (Taq Pol) and Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), in proportions and under reaction conditions that allow one-step amplification of a nucleic acid of interest. In particular, the compositions are useful in one-step RT-qPCR and RT-LAMP methods. The method includes sampling the specimen from a subject, extracting and purifying RNA from the sample. The compositions and methods may be used to produce, analyze, quantitate, detect and otherwise manipulate nucleic acid molecules. For example, disclosed are methods of detecting the presence of a viral nucleic acid in a sample from a subject. Methods of diagnosing a subject as being infected with a viral pathogen are also provided. A preferred virus or pathogen is SARS-CoV-2.

Abstract: Described are affinity purification system that includes a carrier/surface that is non-cellular, and sliding clamp (SC) protein, and methods for purifying proteins that bind to the SC. The SC is associated with the carrier/surface via covalent/non-covalent interactions. To attain control of coupling site, the SC can be mutated via site-directed mutagenesis to introduce an exogenous residue and, the exogenous internal residue is conjugated to the non-cellular surface through the linker. The SC can also be coupled to the carrier via non-covalent interactions such as the affinity interactions involved in ligand/binding partner complex formation. The SC-based affinity purification system are used in a purification column as bait proteins, to isolate SC binding partners or non SC-binding proteins engineered to contain a SC binding site prior to its purification.

Abstract: Provided are methods for recombinantly producing enzymatically active glycosyltransferase (GT) enzymes. Active recombinant glycosyltransferase enzymes and method of use thereof are also provided. The methods for recombinantly producing enzymatically active GTs relies on a yeast expression system, preferably, a Pichia pastoris, expression system and more preferably, a Pichia pastoris stain with an ade2 deletion. Recombinantly produced enzymatically active GT enzymes produced according to the methods disclosed herein can be used for cell surface glycan engineering. The method includes contacting a cell with the disclosed compositions comprising purified recombinant GT enzyme and a substrate (nucleotide sugar) for the GT enzyme for an effective time for the GT enzyme to catalyze transfer of its substrate onto an acceptor site at the surface of the cell. The composition in preferred embodiments does not include glycerol as a stabilizer or it includes at least 50% glycerol.