| My dissertation work focused on the study and manipulation of inteins for their application in biotechnology. I helped genetically define the protein-splicing domain of the RecA intein from Mycobacterium tuberculosis (Mtu) and effectively reduced its size from 48 kDa to 18 kDa. This size reduction supports the model that canonical inteins result from the invasion of a primordial mini-intein gene by an endonuclease gene. I also identified three new inteins from different mycobacterial species, and they are consistent with the “homing cycle model” of endonuclease maintenance.; Through random mutagenesis followed by selection with rationally designed genetic systems, we obtained two mini-intein derivatives with enhanced protein-splicing and C-terminal cleavage activities, respectively. One is termed the splicing mutant (ΔI-SM), while the other is called the cleavage mutant (ΔI-CM). I discovered that the C-terminal cleavage activities of these derivatives are pH-sensitive, being much faster at pH 6.0 than at pH 8.5. We then developed a generic protein purification system based on this property. The ΔI-CM mini-intein is fused to an affinity tag at its N-terminus and a target protein at its C-terminus. The fusion precursor was overexpressed and purified by affinity chromatography, and pH shift was used to trigger the C-terminal intein cleavage to release the target protein. I also identified a mutation that can reverse the pH-sensitivity of ΔI-CM. This mutant can potentially adapt our system to protein expression in a low-pH environment such as transgenic milk.; I also developed a novel intein-mediated approach to purify cytotoxic proteins. The idea involves insertional inactivation of the target protein by an intein, followed by pH-controllable in vitro splicing to restore its configuration and function. The cytotoxic homing endonuclease I-TevI has been successfully purified with this approach.; Finally, I scaled the intein-mediated protein purification systems down 200-fold in a microfluidic platform. This microfluidic system has the potential to be used in multiple parallel separations for various proteomics applications. |
