Studies on recombinant fluorescent macromolecules: Expression, cultivation and purification, with applications towards the isolation of nucleic acids.

摘要

These studies focus on fluorescent proteins and culminate with the production of a synthetic gene for expressing a fluorescent protein. Initial studies led to the publication of technical notes (see appendix) describing the quantitation of green-fluorescent protein (GFP) both in whole bacteria and in solution. These led to studies with large-scale expression, cultivation, and purification of recombinant GFP showcasing various techniques including large-scale fermentation and chromatography, culminating in a paper in Methods in Enzymology, Bioluminescence and Chemiluminescence, Part C. 305:212-23, 2000. These large-scale techniques were needed to overcome poor expression and inefficient purification methods in order to produce enough protein for study. Another study, with a mutant variant of GFP called enhanced green-fluorescent protein or eGFP, was used to explore mutagenesis of the GFP gene as well as new and more efficient approaches to expression and purification such as the application of non-lytic methods of extracting macromolecules. Yet another study applies this experience with the non-lytic method of extraction to isolate plasmid DNA. Finally, a synthetic gene was generated to express a protein based solely on its known primary structure. This protein, a red-fluorescent protein (RFP), described originally from a non-bioluminescent organism, became a platform for exploring techniques in synthetic gene design and creation. It also allowed for pursuing different methods of expressing and purifying fluorescent proteins in a recombinant system. These methods include an aggressive method of osmotic shock to produce on extract that is relatively free of endogenous proteins, as well as a technique for batch precipitation of protein based on the histidine tag which allowed for very specific precipitation of recombinant protein.