Novel Split-Intein Based Protein Trans-Splicing System And Its Applications

Inteins are intervening proteins within precursor proteins and can be viewed as the protein equivalent of introns. Inteins catalyze a protein splicing reaction that excises the intein sequence from the precursor protein and joins the flanking sequences (N-and C-exteins) with a peptide bond. In case of split-inteins, the intein consists of two parts (fragments) that are on two separate precursor proteins expressed from two separate genes. An N-precursor protein contains an N-extein followed by the intein’s N-terminal fragment (IN), while a C-precursor protein contains the inteins’s C-terminal fragment (IC) followed by a C-extein.IN and Ic on the two precursor proteins can reassemble non-covalently to catalyze a trans-splicing reaction that joins the two exteins. Protein trans-spling has become a powerful tool of protein engineering, which includes fusion of separate polypeptides, gene therapy, etc.For conventional (SO form) split-inteins, In and Ic correspond to a split of the intein sequence at a split site near the middle of the intein, therefore both IN and Ic of SO split-intein have relatively large sizes. Recently, novel split-inteins (S1form) have been engineered by splitting the intein sequence near the N-terminus of the intein, resulting in a small IN that is11amino acids (aa) long. When the N-extein (EN) is also small, the N-precursor protein (EN-IN) can be produced as a synthetic peptide. The N-extein on this peptide can be chemically modified or labeled in the process of peptide synthesis, then this site-specially modifier (or labeled) N-extein can be added to a target protein of interest (C-extein) through protein trans-splicing. This method has been demonstrated successfully in N-terminal fluorescent labeling of recombinant proteins in vitro and on mammalian cell surface, using Ssp DnaB S1split-intein. In contrast, a novel S11split-intein has been engineered to have a6-aa IC, so that a synthetic peptide could be made to contain this small IC and a small C-extein carrying desired chemical groups, and this has been shown to be useful in adding fluorescent groups to the C-terminus of recombinant target proteins, using Ssp GyrB S11split-intein. Fluorescent or isotope labels can be useful for studying cellular location and trafficking of proteins, and chemical modifications (e.g. unnatural amino acids) can aid study of protein structure-function relationship. Standard chemical methods often produce mixed populations of the modified protein, because such methods usually target certain amino acid side chains (e.g. thiols, carboxyls, amines) that may exist at multiple locations in the protein. Other methods have been developed for site-specific protein modifications with limited success. Intein-based protein-peptide trans-splicing is a newer and potentially more useful method for protein modifications, because it is site-specific and does not leave a large tag in the modified protein and may be used generally with any chemical moieties on the chemically synthesized extein peptide. To further develop this intein-based method, it is important to find new and atypical split-inteins for the peptide-protein trans-splicing, as different inteins may exhibit different splicing efficiencies with different exteins. When searching for new atypical split inteins, we noticed that a large I(?) derived from an Ssp DnaX intern was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. To address these questions, we show here that this IC could undergo trrans-splicing in the presence of IN, and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this IC could trans-splice with small IN sequences derived from two other inteins, showing a cross-reactivity of this atypical split-intein. Furthermore, we found that this IC could trans-splice even when the IN sequence was embedded in a nearly complete intein sequence, suggesting that the small IN could project out of the central pocket of the intein to become accessible to the IC Overall, these findings produced a new atypical split-intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.In studying the Ssp GyrB intein that had been converted into an atypical SIl split-intein previously, we found that this intein could also be converted into SI split-intein, and trans-splicing could be achieved in a form of Sl-Sl1overlapping split intein. This finding adds significantly to the toolbox of intein-based technologies, particularly for joining recombinant proteins through trans-splicing. Previously the SO form split-inteins have been used the most, however one or both of its intein pieces often caused misfolding or insolubility of the intein-containing fusion proteins. This was thought to be due to the SO split site that is near the middle of the intein sequence and more likely to cause large disruptions of the single-domain structure of the intein. In contrast, our overlapping split-intein was derived from the SI and SII split sites near the two termini of the intein sequence, so that each of the two larger intein fragments is almost an intact intein and less likely to misfold. Another useful feature of our new split-inteins is that an hexahistidine tag could be added in the intein sequence without inhibiting the splicing activity, which permits easy purification of the intein-containing precursor proteins through affinity binding to commonly used nickel beads.Additionally, we evaluated C-inteins of two additional inteins, Rma DnaB and Ssp GyrB S1split-inteins, for cross-reactivity with N-inteins of different S1split-inteins, in order to determine whether S1split-inteins are generally promiscuous. We found that the C-intein of Rma DnaB S1split-intein could cross-react with the N-intein of Ssp GyrB S1split-intein, but not with the N-intein of Ssp DnaX SI split-intein. Similarly, the C-intein of Ssp GyrB S1split-intein could cross-react with the N-intein of Rma DnaB S1split-intein, but not with the N-intein of Ssp DnaX S1split-intein. It indicates that promiscuity is a general phenomenon among S1split-inteins and not restricted to the previously reported C-intein of the.Ssp; DnaX S1split-intein. In addition, some of the cross-reactivities also showed a temperature-dependence.We then did a more systematic study of cross-reactivity between different S1or S11split-inteins by constructing a large number of atypical hybrid split-inteins followed by analysis of their in vitr trans-splicing activities. As a result, we got many S1and S11split-inteins that can splice specifically without cross-reactivity. A new database was thereby established including combinations of S1split-inteins, S11split-inteins, or S1&S11split-inteins those could splice specifically. Co-splicing of these split-inteins in one system will be quite useful for the application of novel split-inteins in protein research and engineering. Co-existence of two or more split-inteins in one system (or test tube), without cross-reactivity, can be quite useful for the application of split-inteins in protein research and engineering. For example, different proteins may be specifically modified or labeled using specific split-inteins in a cell or a test tube containing multiple proteins. We have demonstrated this method using model proteins in a test tube, using several pairs of non-cross reacting split-inteins including SI split-inteins, S11split-inteins, or S1&S11split-inteins. Another use of the non-cross reacting S1&S11split-inteins is internal peptide splicing, in which a small peptide can be inserted into the target protein. Out of9pairs of S11&S1split-inteins tested, eight pairs successfully inserted a peptide into a model protein (exteins). In these reactions, the middle precursor is quite small and may be chemically synthesized with desired modifications or labels, therefore this internal peptide splicing method may be used to modify or label proteins at internal sites.The above protein trans-splicing systems we have established may also be used in many other areas. For example, if a cyto-toxic protein cannot be expressed in a cell due to toxicity, the protein may be expressed as two non-toxic fragments that can subsequently be spliced together to produce the complete protein. If a large protein cannot be expressed due to its size, it may be expressed in several smaller parts and sequentially ligated through the splicing of multiple split-inteins. Site-specific fluorescence labeling of multiple proteins may be carried out using the multiple split-inteins that do not cross-react. This may be useful for fluorescence resonance energy transfer (FRET) that is an important technique in the research of large biological molecules interactions, cell physiology, immunoassay, etc. Ou trans-splicing system may also be used to introduce segmental isotope into proteins for Nuclear Magnetic Resonance (NMR) studies.In this thesis we focus on engineering of novel split inteins, intein structure-function research, cross reactivity among different split inteins, establishment of a new trans-splicing system using multiple split inteins and its applications. Our findings may help people have more understanding of inteins, provide more methods in protein research and engineering, improve the development of biotechnology, biomedicine and protein chemistry.