Rational design and directed evolution of novel streptavidin and intein mutants for biotechnology applications

In the first of two sections of this dissertation, we discuss recent advances with engineered monomeric streptavidin. Streptavidin is widely used in biotechnology because of its high affinity interaction with the small molecule biotin. However, the tetrameric state of wild type streptavidin precludes its use in applications where target aggregation is a concern. To further extend the use of the streptavidin-biotin technology, our group recently engineered a streptavidin monomer, mSA, which possesses low nanomolar affinity for biotin. The designed monomer has a dissociation half-life (t½) of 83 minutes at 25°C, which is the slowest biotin dissociation kinetics reported for a streptavidin monomer, but slower dissociation kinetics would allow more stable labeling of target molecules. Through targeted mutations based on the bradavidinII sequence, we have engineered streptavidin monomers with ~50 fold improvement in t½ compared to first generation mSA.;An important technological advancement that accompanied the design of new mutants is a secretion method that facilitates purification of mSA from bacteria. When expressed alone (i.e., without the aid of a solubility tag) mSA is insoluble and appears exclusively in inclusion bodies which created a serious roadblock in the practical adoption of mSA in biotechnology. Initially, the purification procedure lasted roughly one week and resulted in yields that harbored around 3 mg per 1 L of bacterial culture. We subsequently developed a simpler purification protocol by fusing mSA to either maltose binding protein or thioredoxin. Because the fusion protein remained in the soluble portion of the cell lysate, the refolding step was no longer needed. If necessary, the fusion tag can be removed using an internal tobacco etch virus (TEV) protease recognition sequence. Although cumbersome due to several rounds of chromatography steps, the fusion tag method can yield > 100 mg of purified mSA from 1 L of culture. We recently developed a new purification protocol based on the Sec-dependent secretion pathway in E. coli. Through incorporation of an OmpA signal, mSA is secreted to the culture medium, from which the molecule can be isolated using a hexahistidine affinity column. Because mSA is recovered without cell lysis, the purity of the protein is higher than that achieved through intracellular expression and only one round of purification is required. Furthermore, since the OmpA signal is proteolytically cleaved during transport through the inner membrane, a separate TEV protease cleavage step is avoided. Taken together this process is a quick, efficient, and can generate high yields of pure mSA.;In the second section of this dissertation, we discuss recent progress with the trans-splicing inteins DnaE and DnaB. Inteins are protein segments embedded in-frame within a precursor sequence that catalyze a self-excision reaction and ligate the flanking sequences (exteins) with a standard peptide bond. A small fraction of inteins are expressed as two separate fragments, and trans-splice upon subunit association. These split inteins have found widespread use in a variety of biotechnology applications, but domain assembly is currently constrained to two exteins, and attempts to use split inteins to assemble additional domains within the cell remains a challenge. Through orthogonally acting split inteins DnaE and DnaB, we show the three domain assembly of a 130 kDa protein MBP-eGFP-tdTomato within E. coli. This technique allows facile assembly of multi-domain proteins, overcoming certain barriers associated with protein solubility and size that limit protein production in E. coli. It serves as a stepping stone for the advancement of intein-mediated protein synthesis, which has a potentially significant impact in biotechnology.;In the final two chapters, we report on the development of a temperature sensitive trans splicing DnaE intein. Temperature sensitive mutants represent a class of conditional mutants that lose activity at an elevated ambient temperature. These mutants are useful in elucidating the function of a protein but difficult to engineer systematically. Because intein splicing can occur independently of extein context, Ts inteins can be used to conditionally control a variety of proteins, circumventing the labor-intensive task of identify Ts mutants on an individual protein-of-interest basis. To this end, we developed a survival assay based on the subcellular localization of the reverse tetracycline-controlled transcriptional activator (rtTA) and used the assay to identify temperature-sensitive trans-splicing inteins. Through the use of nuclear localization signal (NLS) and nuclear export signal (NES), rtTA translocation into the nucleus can be controlled based on successful intein splicing. If nuclear rtTA drives the transcription of URA3, the growth of the uracil auxotroph yeast strain is dependent on the activity of the split intein. Replica plating was used to identify ts variants of DnaE intein from Nostoc punctiforme PCC73102 (Npu), which splices efficiently at 20°C but not at 37°C. (Abstract shortened by UMI.).