| Intein is an internal protein sequence that can excise itself from the premature protein and ligate the flanking sequences by a native peptide bond. Normally, the protein splicing reaction can be finished in four steps by the conserved residues, and mutation at the conserved residues can convert the splicing intein into a cleaving element For example, mutation of the first conserved Cys to Ala results in a C-terminal cleaving intein. Based on its splicing and cleaving functions, intein has many potential applications in protein engineering, such as for protein purification, as a molecular switch, in biosensors, or for protein labeling. In this thesis, we will focus on the protein purification and biosensor applications.Combining the cleaving intein with a protein purification tag, it results in a self-cleaving tag. Hundreds of proteins have been purified by self-cleaving tags in the last twenty years, and the used fusion tags include normal chromatographic tags and novel non-chromatographic tags. However, there are still several bottleneck problems in the existent system, including intein premature cleaving in vivo during expression and the low cleaving efficiency for the specific protein. In particular, based on different expression conditions the premature cleaving efficiency is from10-80%. Premature cleaving is especially a problem in the eukaryotic host cells where there is no detectable full length precursor. As protein splicing, intein cleaving behavior also shows a target protein preference. When fused with green fluorescence protein (GFP), the cleaving efficiency of the Mtu RecA△I-CM is only60%after incubation at room temperature in a low pH cleaving buffer. These problems will hamper the wide usage of intein technology in production of recombinant protein.To reduce the premature cleaving in vivo, we applied the split intein systems and Zinc sensitive mutant inteins. Because of the deficit in the structure, normally, the incomplete intein does not have activity, and supplementing the complementary element can restore intein activity. Here we split the AI-CM intein into two fragments and each of them was fused with a non-chromatographic ELP tag. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein-ELP system. Further, a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, shows higher cleaving efficiency and allows the segments to be co-purified in a single reaction mixture. The other split intein system is based on the S1split Ssp DnaB intein. The first11amino acids are removed from the full length intein and the remaining intein fragment is inserted into the ELP-intein system by replacement of the original AI-CM. The precursor without the first11amino acids is not able to cleave in vivo during expression; however, with addition of the complementary small (11amino acid) peptide in vitro the intein cleaving activity can be restored. Applying this system we have purified several well studied module proteins, including P-lactamase, enhanced green fluorescence protein and maltose binding protein.During the intein crystallization, a zinc binding domain was found which was close to the C-terminal function domain. Later study showed that zinc can inhibit the protein slicing and N-terminal cleaving, and the inhibition was reversible by addition of metal-chelator (EDTA). While the C-terminal cleaving reaction is not as sensitive to the zinc as the intein splicing or N-terminal cleaving. The in vitro binding assay showed that the low zinc binding with conserved residues for the C-terminal cleaving could account for this. Based on the known structure information, we introduced several mutations at intein N-terminal context which consists of a zinc binding domain with the C-terminal cleaving key residue His439. Here, we designed six zinc sensitive candidate mutants (001,002,003,004,03b and04b), and these mutants were assayed in a well-studied intein activity assessment system, MBP-Intein-aFGF. The premature cleaving of one of these mutants (002) was too fast to get a full length precursor and one of the mutants (001) did not show the cleaving activity both in vivo and in vitro. Other mutants, including the wild type AI-CM, showed sensitivity to the zinc and inhibition efficiencies of the C-terminal cleaving were higher than80%in the presence of1mM ZnC12, and intein activity could be recovered by supplementing with EDTA. To quantify the zinc inhibition efficiency, the EC50(half effective concentration) was calculated from the dose-response. Except003which has similar ECso to the wild type, the other mutants all showed advantages over the wild type. In particular, EC50of004was half of the wild type and the EC50S of03b and04b were only one-fifth of the wild type. Further, the C-terminal cleaving of more zinc sensitive mutants (03b and04b) was significantly faster than the wild type.As in protein splicing, the cleaving intein also showed a preference of the target protein, which would also affect the application of this technology. Here we made a mutation pool with the natural20amino acids at the intein first residue, and after screening a more general and faster C-terminal cleaving mutant (IG) which started with Gly was found. Further, the IG showed to be more sensitive to the environmental temperature. In particular, the IG could complete the C-terminal cleaving in5h when fused with GFP, while the cleaving efficiency from the wild type was only-70%under the same condition. And there was not much difference in the cleaving rate when assayed at room temperature. To our surprise, we found an amide bond between intein’s last Asn and-1Lys or-3Lys, but main side reaction products were found from the-1site (over90%). Based on this side reaction, we made a recombinant cyclized lactamase protein which also showed equivalent thermo stability to normal cyclized protein made by other ways.Besides the application in protein purification, intein also can be applied to hormone biosensors based on its ability to stabilize the structure of fusion protein. We previously have successfully developed several hormone bacterial sensors, including estrogen and thyroid. This system is based on a cell growth phenotype assay. There are several advantages of the bacterial biosensor system when comparing to the existent animal system, including low cost and high throughput. Here we introduced six clinically relevant mutations which can result in resistance to thyroid hormone (RTH) into our human thyroid receptor beta (TRp) biosensor and examined the resulting binding effects on recognized native and synthetic TR ligands. Our results indicate that these TRP mutations are not equal in their effects on ligand binding, and that both agonists and antagonist binding are highly dependent on each specific mutation. Some mutations lead to partial loss of binding for some ligands (EC50values increase from several to hundred times), while other mutants completely lost binding ability for all ligands. Furthermore, we found two mutant sensors are more sensitive to the antagonist, which may indicate the mutation may interrupt the competing agonist binding.In conclusion, here we optimized intein technology for protein purification and also applied the bacterial biosensors based on intein to study six clinical thyroid hormone mutations. This work can provide important information to intein cleaving mechanism and also can expand the intein technology in protein purification, especially in eukaryotic host cells which normally produce the more complicated functional proteins. Importantly, the bacterial hormone biosensor which is based on an intein fusion protein can allow rapid prototyping of new mutant assay systems, with the goal of accelerating the evaluation of relatively safe thyromimetics for possible supplementation or rescue mode applications in early diagnosis RTH patients. |
