Patent Application
20160319287
Inteins are internal protein sequences that excise themselves out of a precursor protein in an autocatalytic reaction called protein splicing. In protein trans-splicing the intein domain is split and located on two separate polypeptides. Protein trans-splicing catalysed by split inteins is a powerful technique to assemble a polypeptide backbone from two separate parts. During the reaction the N- and C-terminal intein fragments (also termed IntN and IntC) first associate and fold into the active intein domain and then link the flanking sequences, also termed the N- and C-terminal exteins (ExtN and ExC), with a peptide bond while at the same time precisely excising the intein sequence. Apart of their homologous N- and C-terminal exteins, inteins will generally also excise themselves out of heterologous sequence flanks. Moreover, an intein is a self-contained entity, that is, it does not require any additional cofactors or energy sources to perform the protein splicing reaction.
The split intein based trans-splicing reaction has found various applications in basic protein research and biotechnology, e.g. for segmental isotope labelling of proteins, preparation of cyclic polypeptides, transgene expression, as well as more recently for chemical modification of proteins and protein semi-synthesis (R. Borra, J. A. Camarero, Biopolymers 2013; T. C. Evans, Jr., M. Q. Xu, S. Pradhan, Annu. Rev. Plant Biol. 2005, 56, 375; C. J. Noren, J. Wang, F. B. Perler, Angew. Chem. 2000, 112, 458; Angew. Chem. Int. Ed. Engl. 2000, 39, 450; M. Vila-Perello, T. W. Muir, Cell 2010, 143, 191-200; G. Volkmann, H. Iwai, Mol. Biosyst. 2010, 6, 2110; G. Volkmann, H. D. Mootz, Cell. Mol. Life. Sci. 2013, 70, 118).
However, split inteins are rare, and especially for chemical modification of proteins and protein semi-synthesis special properties are required. Specifically, one of the intein fragments should be as short as possible to facilitate its efficient and inexpensive assembly by solid-phase peptide synthesis. All naturally occurring split inteins reported so far show the break-point at the position of the homing endonuclease domain in the related contiguous maxi-inteins. This split site gives rise to an IntN of about 100 amino acids (aa) and an IntC of about 35 aa (I. Giriat, T. W. Muir, J. Am. Chem. Soc. 2003, 125, 7180-7181; H. Wu, Z. Hu, X. Q. Liu, Proc. Natl. Acad. Sci. USA 1998, 95, 9226). Split inteins with shorter IntN or IntC fragments have been created artificially from naturally contiguous inteins (J. H. Appleby, K. Zhou, G. Volkmann, X. Q. Liu, J. Biol. Chem. 2009, 284, 6194; A. S. Aranko, S. Zuger, E. Buchinger, H. lwai, PloS One 2009, 4, e5185; Y. T. Lee, T. H. Su, W. C. Lo, P. C. Lyu, S. C. Sue, PloS One 2012, 7, e43820; C. Ludwig, M. Pfeiff, U. Linne, H. D. Mootz, Angew. Chem. 2006, 118, 5343; Angew. Chem. Int. Ed. Engl. 2006, 45, 5218; W. Sun, J. Yang, X. Q. Liu, J. Biol. Chem. 2004, 279, 35281; G. Volkmann, X. Q. Liu, PloS One 2009, 4, e8381), but these generally show lower splicing yields and rates and tend to associate and fold less efficiently. Moreover, another drawback of known split inteins is a limited compatibility with diverse target proteins due to the solubility and expression issues of the recombinant split intein fusion constructs.
Hence, there exists need in the art for alternative split inteins that ameliorate or overcome the known problems.
The present invention is based on the unexpected finding that specific inteins or fragments of said inteins have the property that the N-terminal intein fragment of said intein is split after only 14-60 amino acids from the intein's N-terminal end, with these inteins being naturally split inteins. These inteins provide for the shortest naturally occurring N-terminal intein fragments discovered so far. Moreover, they exhibit excellent splicing yields and rates.
Thus, in a first aspect, the present invention relates to an isolated polypeptide comprising at least one intein or at least one fragment of said intein, wherein said intein is a naturally split intein with a N-terminal intein fragment split after 14-60 amino acids from the intein's N-terminal end.
Due to the very short N-terminal parts, these inteins or their IntN fragments, respectively, can be easily generated via solid peptide synthesis, which is faster, more reliable and robust than protein generation via recombinant protein expression techniques.
Hence, these novel split inteins are ideally suited for all kinds of efficient protein modifications.
Moreover, it was surprisingly found possible to further modify some of these natural split inteins to even increase splicing yields and rates and thus render the novel split inteins suited for an even wider range of applications and assay conditions.
In various aspects, the present invention relates to an isolated polypeptide as described above wherein said at least one intein or intein fragment is selected from the group comprising:
In various further aspects, the present invention relates to an isolated polypeptide as described above, wherein said polypeptide comprises at least one N-terminal intein fragment and at least one C-terminal intein fragment, wherein
In various further aspects, the present invention relates to an isolated polypeptide as described above wherein said polypeptide at the N-terminal end of the at least one N-terminal intein fragment and/or at the C-terminal end of the at least one C-terminal intein fragment further comprises a flanking amino acid sequence, wherein said flanking amino acid sequence is selected from:
In a further aspect, the present invention relates to an isolated polypeptide as described above, wherein the N-terminal intein fragment has (or comprises an amino acid sequence that has) at least 90%, at least 95% or 100% amino acid sequence identity with an amino acid sequence as set forth in any one of SEQ ID Nos. 5, 11 and 12, and/or wherein the C-terminal intein fragment has (or comprises an amino acid sequence that has) at least 90%, at least 95% or 100% amino acid sequence identity with an amino acid sequence set forth in any one of SEQ ID Nos. 6 and 13-18.
In a still further aspect, the invention relates to an isolated polypeptide as described above, wherein the N-terminal intein fragment has an amino acid sequence comprising or consisting of any one of the amino acid sequences set forth in SEQ ID Nos. 5, 11 and 12 and/or, wherein the C-terminal intein fragment has an amino acid sequence comprising or consisting of any one of the amino acid sequences set forth in SEQ ID Nos. 6 and 13-18.
In various further aspects, the invention also encompasses an isolated polypeptide as described above, wherein the polypeptide comprises an N-terminal intein fragment having the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:12 and a C-terminal intein fragment having the amino acid sequence set forth in SEQ ID NO:13.
In still other aspects, the invention relates to an isolated polypeptide as described above, wherein the N-terminal intein fragment has at least 90%, at least 95% or 100% amino acid sequence identity with an amino acid sequence as set forth in any one of SEQ ID Nos. 5, 28-33, and/or wherein the C-terminal intein fragment has at least 90%, at least 95% or 100% amino acid sequence identity with an amino acid sequence as set forth in SEQ ID NO:27.
In a further aspect, the present invention relates to an isolated polypeptide as described above, wherein the polypeptide further comprises at least one C-terminal extein and/or at least one N-terminal extein sequence.
In a further aspect, the present invention relates to two isolated polypeptides as described above or a combination of two polypeptides as described above or a composition comprising those, wherein the first isolated polypeptide comprises at least one heterologous N-terminal extein fused to an N-terminal intein fragment having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID Nos. 5, 28, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176 and 182, and wherein the second isolated polypeptide comprises at least one C-terminal extein sequence fused to a C-terminal intein fragment having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of the amino acid sequences set forth in SEQ ID Nos. 6, 27, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177 and 183.
In a further aspect, the present invention relates to an isolated polypeptide as described above, wherein the polypeptide or any one of the two polypeptides further comprises at least one component selected from a solubility factor, a marker, a linker, an epitope, an affinity tag, a fluorophore or a fluorescent protein, a toxic compound or protein and a small-molecule or a small-molecule binding protein.
In a further aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding for at least one polypeptide as described herein or a homolog, variant or complement thereof.
In a further aspect, the present invention relates to a method using an isolated polypeptide or a nucleic acid molecule as described above, wherein the method is selected from the group consisting of modification of a protein, protein lipidation, protein immobilization, protein backbone semi-synthesis, regioselective protein side chain modification and artificial control of protein splicing by light.
In a further aspect, the present invention relates to the use of a polypeptide or a nucleic acid molecule according as described above, wherein the use is selected from the group consisting of modification of a protein, protein lipidation, protein immobilization, protein backbone semisynthesis and use as molecular switch.
In a further aspect, the present invention relates to a kit comprising at least one polypeptide or a nucleic acid molecule as described above.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.
In
Top: Rate constants and product yields of the AceL-TerL intein (SEQ ID NO:1)
Bottom: Rate constants and product yields of the mutants M1-M6.
The results presented in
Splicing with constructs containing the POIs
As stated above, the present invention is based on the unexpected finding of novel naturally split inteins that split after 14-60 amino acids from the intein's N-terminal position. “N-terminal position”, as used in this context, refers to the numbering starting from the utmost N-terminal amino acid, which is assigned position number 1. These inteins provide the shortest naturally occurring N-terminal intein fragments discovered so far. Moreover, they exhibit excellent splicing yields and rates.
In a first aspect the present invention thus relates to an isolated polypeptide comprising at least one intein or at least one fragment of said intein, wherein said intein is a naturally split intein with a N-terminal intein fragment split after 14-60 amino acids from the intein's N-terminal end.
As used herein, the term “isolated polypeptide” refers to a polypeptide, peptide or protein segment or fragment, which has been separated from other cellular components with which it may naturally associate and, in certain embodiments, which has been excised out of sequences, which flank it in a naturally occurring state. In other words, the isolated polypeptide may be a polypeptide fragment, which has been excised from a longer polypeptide sequences, in particular sequences which are normally adjacent to the fragment in the naturally occurring protein. As such, the isolated polypeptides may be artificial polypeptides. As mentioned above, the term is also used here to designate a polypeptide, which has been substantially purified from other components, which naturally accompany the polypeptide, e.g., proteins, RNA or DNA which naturally accompany it in the cell. The term therefore includes, for example, a recombinant polypeptide, which is encoded by a nucleic acid incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant polypeptide, which is part of a hybrid polypeptide comprising additional amino acids.
Moreover, the isolated polypeptide described herein or the nucleic acid encoding it may comprise in addition to all features described below regulatory sequences, i.e. segments that on nucleic acid level are capable of increasing or decreasing the expression of specific genes within an organism or segments that on protein level regulate posttranslational processing, cellular localization and the like.
The term “intein” as used herein refers to a segment of a protein capable of catalysing a protein splicing reaction that excises the intein sequence from a precursor protein and joins the flanking sequences (N- and C-exteins) with a peptide bond. Hundreds of intein and intein-like sequences have been found in a wide variety of organisms and proteins. They are typically 150-550 amino acids in size and may also contain a homing endonuclease domain.
The term “split intein” as used herein refers to any intein, in which one or more peptide bond breaks exists between the N-terminal and C-terminal amino acid sequences such that the N-terminal and C-terminal sequences become separate fragments that can non-covalently reassociate, or reconstitute, into an intein that is functional for trans-splicing reactions. In other word, a split intein, is an intein consisting of two separate polypeptides that can non-covalently associate to perform the intein function, with one of said polypeptides comprising the N-terminal part and the other comprising the C-terminal part. In case the respective polypeptides are coupled to exteins, these exteins are covalently linked by said association of the intein parts.
The term “intein fragment” as used herein refers to a separate molecule resulting from peptide bond breaks between the N-terminal and C-terminal amino acid sequences in (split) inteins. In other words, the term “intein fragment”, as used herein, relates to one of the separate parts of a split intein, in particular either the N-terminal or the C-terminal part. Such a fragment can associate with its counterpart fragment to form the active split integrin.
As the N-terminal intein fragments of the inteins described herein are comparably short, the isolated polypeptides are ideally suited for use over a wide range of protein modification techniques, such as modification of therapeutic proteins, since the protein of interest-IntN (POI-IntN) peptide complex or, more generally, the modifying moiety-IntN peptide complex can be easily obtained using solid-phase peptide synthesis and, optionally, synthetic chemistry. Moreover, since these inteins are natural split inteins generated by evolution, they exhibit high splicing yields and rates, without exhibiting the problems encountered with split inteins artificially engineered to have short IntN fragments.
In a preferred embodiment of this aspect of the present invention the N-terminal intein fragment is split after 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids as calculated from the intein's N-terminal end. In an especially preferred embodiment the N-terminal intein fragment is split after 24, 25 or 36 amino acids from the intein's N-terminal end.
In various embodiments of this aspect of the present invention the intein is a naturally split intein with a N-terminal intein fragment split after 24-37 amino acids from the intein's N-terminal position. Thus, the N-terminal intein fragment of such an intein and/or the protein of interest-IntN peptide complex is even shorter and hence better suited for the chemical synthesis, e.g. for solid peptide synthesis, which is faster, easier to perform and much more reliable than protein generation via recombinant protein expression.
In a further aspect the present invention relates to an isolated polypeptide as described above, wherein said at least one intein fragment, is selected from the group comprising:
In a preferred embodiment of the invention, the split intein is formed by two separate polypeptides that non-covalently associate, i.e. there is one C-terminal intein fragment and one N-terminal intein fragment.
As interchangeably used herein, the terms “N-terminal split intein, “N-terminal intein fragment” and “N-terminal intein sequence” (abbreviated “IntN”)” refer to any intein sequence that comprises an N-terminal amino acid sequence that is functional for trans-splicing reactions. It thus also comprises a sequence that is spliced out when trans-splicing occurs. It can comprise a sequence that is a modification of the N-terminal portion of a naturally occurring intein sequence. For example, it can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the IntN non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the IntN.
As interchangeably used herein, the terms “C-terminal split intein”, “C-terminal intein fragment” and “C-terminal intein sequence” (abbreviated “IntC”)” refer to any intein sequence that comprises a C-terminal amino acid sequence that is functional for trans-splicing reactions. An IntC thus also comprises a sequence that is spliced out when trans-splicing occurs. An IntC can comprise a sequence that is a modification of the C-terminal portion of a naturally occurring intein sequence. For example, it can comprise additional amino acid residues and/or mutated residues so long as the inclusion of such additional and/or mutated residues does not render the IntC non-functional in trans-splicing. Preferably, the inclusion of the additional and/or mutated residues improves or enhances the trans-splicing activity of the IntC.
The term “sequence identity” as used herein refers to peptides that share identical amino acids at corresponding positions or nucleic acids sharing identical nucleotides at corresponding positions. In order to take into account the fact that peptides may exist which do not have significant “sequence identity”, as they may not have similar amino acids at corresponding positions, but have the same function, because they contain, e.g., conservative substitutions, the amino acid sequences herein are referred to in the context of percent identity.
The determination of percent identity described herein between two amino acid or nucleotide sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) world wide web site having the universal resource locator “www.ncbi.nlm.nih.gov/BLAST”. Blast nucleotide searches can be performed with BLASTN program, whereas BLAST protein searches can be performed with BLASTX program or the NCBI “blastp” program.
The term “mutant” as used herein refers to polypeptide the sequence of which has one or more amino acids added, deleted, substituted or otherwise chemically modified in comparison to a reference polypeptide, for example one of the claimed sequences, provided that the mutant retains substantially the same properties as the reference polypeptide. “Substantially the same properties”, in various embodiments, relates to the fact that a given mutant has at least 50%, preferably at least 75% or more of the activity of the reference polypeptide.
In various embodiments, the isolated polypeptide comprises at least an N-terminal intein fragment having at least 70%, or 80% or 85% or 90% or 95% or 100% sequence identity with the amino acid sequence set forth in any one of SEQ ID Nos. 2, 3, 4, 5, 28, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176 and 182.
In various embodiments, the isolated polypeptide comprises at least a C-terminal intein fragment having at least 70%, or 80% or 85% or 90% or 95% or 100% sequence identity with the amino acid sequence set forth in any one of SEQ ID Nos. 6, 27, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 194, 195 or 196.
To form the functional split intein, the polypeptide comprising at least one N-terminal intein fragment as defined above and the polypeptide comprising at least one C-terminal intein fragment may be combined. It is understood that the functional split intein is formed, in various embodiments, by two of the isolated polypeptides described herein, one comprising the N-terminal and the other the C-terminal part, with both being separate molecules, i.e. not being covalently linked by a peptide bond.
The isolated polypeptides described herein can advantageously be used for example for labelling of a protein. Due to the small size of the N-terminal intein fragment the protein of interest-IntN (POI-IntN) peptide complex can be obtained by using solid-phase peptide synthesis. The label e.g. EGFP attached to the IntC fragment (IntC-EGFP) can be generated by recombinant protein expression. Upon combining the two chimeric protein complexes, i.e. POI-IntN and IntC-EGFP, the trans splicing reaction could take place generating POI-EGFP. Of course, also encompassed are all embodiments wherein N- and C-terminal intein fragments are exchanged, i.e. by coupling the label or any other modifying moiety (that need not be a peptide or protein but only needs to be coupled to an amino acid or amino acid oligomer) to the N-terminal intein fragment and synthesizing the protein of interest as a recombinant fusion protein with the C-terminal intein fragment. It is thus understood that while embodiments may be described herein with reference to only one of these possibilities the present invention is intended to also cover the respective counterpart where the two intein fragments are exchanged.
In various embodiments, the two separate intein fragments are useful by themselves. For example, it is possible, to pre-assemble—possibly in form of a kit—the IntC-EGFP fusion protein or merely the IntC fragment, e.g., for easy protein labelling. The ready IntC-EGFP fusion proteins could be then used as soon as a protein of interest is decided upon for easy and robust protein labelling. Of course, the reverse scenario is also possible, where the protein of interest is known and pre-generated fused to the IntN fragment. As soon as it is decided upon which labels should be used the IntC-label fusion proteins could be prepared and protein labelling could be carried out.
Moreover, the EGFP of the fusion protein in this example can of course be readily replaced by any protein of interest or any other non-peptide, non-protein moiety. In case non-peptide, non-protein moieties are used for modification of proteins or any other purpose, these are used in form of conjugates with at least one amino acid or a short peptide sequence to facilitate the covalent linkage with the corresponding other extein part by a peptide bond.
In various embodiments, including the afore-mentioned, it is preferred that the IntC-protein fusion protein, or more generally the intein fragment-protein of interest fusion protein, is generated via recombinant expression.
In various embodiments of this aspect of the invention, one of the intein fragments can be attached to a short PEG linker with a thiol group and then bound to (immobilized on) a maleimido-coated glass surface. Upon addition of the protein of interest fused to the complementary intein fragment and trans-splicing the protein of interest would remain bound to the glass surface. Thus, it is possible to preassemble such a glass surface, e.g. with the IntN fragment, in order to later on immobilize any protein of interest fused to the complementary IntC fragment. Moreover, IntN fragment preassembled in such a fashion could act as capture probe array.
In various embodiments of this aspect of the present invention an isolated polypeptide comprises at least one N-terminal intein fragment and at least one C-terminal intein fragment, wherein:
In these embodiments, the two fragments that naturally occur in form of separate molecules may be combined in one molecule. Alternatively, the two fragments may still be parts of separate molecules. In the latter case, the isolated polypeptide is a combination of at least two, preferably two, isolated polypeptides, one of which comprises the N-terminal intein fragment, as defined above, and the other comprising the C-terminal intein fragment, also as defined above. The present invention therefore also covers combinations of two isolated polypeptides as described herein, wherein an isolated polypeptide comprises at least one N-terminal intein fragment and at least one C-terminal intein fragment, wherein the first polypeptide comprises at least one N-terminal intein fragment and the second polypeptide comprises at least one C-terminal intein fragment, wherein:
Advantageously the resulting polypeptide has split intein activity exhibiting excellent splicing yields and rates.
Furthermore, of course all application envisaged for one of the intein fragments also apply for both together.
In preferred embodiments of this aspect of the present invention the isolated polypeptide comprises exactly one N-terminal intein fragment and exactly one C-terminal intein fragment selected as described above. This similarly applies in case two separate isolated polypeptides are used.
In yet another aspect the present invention relates to an isolated polypeptide as described above, wherein said polypeptide at the N-terminal end of the at least one N-terminal intein fragment and/or at the C-terminal end of the at least one C-terminal intein fragment further comprises a flanking amino acid sequence, wherein said flanking amino acid sequence is selected from:
In various embodiments, the above is to be understood such that, for example, the N-terminal intein fragment of SEQ ID NO:5 is N-terminally flanked by SEQ ID NO: 25, i.e. SEQ ID NO:25 is located N-terminal to SEQ ID NO:5, and the C-terminal fragment of SEQ ID NO:6 is C-terminally flanked by SEQ ID NO:26, i.e. SEQ ID NO:26 is located C-terminal to SEQ ID NO:6.
Such an isolated polypeptide has the advantage that the autocatalytic reaction—the protein splicing—proceeds with higher efficiency, if 1-5 of the wild type extein residues (also termed flanking sequences, i.e. sequences flanking the intein) are present.
If the intein fragments are part of different polypeptides, the respective flanking sequences may be comprised in the respective polypeptide.
In this context, it is emphasized that in general the sequences in this application are shown without the +1 residue following the IntC, as this residue is strictly not part of the intein. However, it should be noted that this residue is usually involved in the intein's activity and forms part of the intein active site.
In case of an isolated polypeptide/isolated polypeptides comprising one N-terminal intein fragment and one C-terminal intein fragment, wherein the N-terminal intein fragment has at least 70%, or 80% or 85% or 90% or 95% or 100% sequence identity with SEQ ID NO:5 and the C-terminal intein fragment has at least 70%, or 80% or 85% or 90% or 95% or 100% sequence identity with SEQ ID NO:6, the resulting intein has an activity maximum at low temperatures such as 8° C. This is ideal to preserve potentially fragile proteins of interest for example in in vitro protein labelling experiments.
Moreover, it was surprisingly possible to further modify the AceL-TerL intein (SEQ ID NO:1) to increase splicing yields and rates even at 37° C.
Thus, in a further aspect the present invention relates to an isolated polypeptide wherein the N-terminal intein fragment has 100% sequence identity with a sequence selected from sequences comprising SEQ ID NO:5, 11 and 12, or has at least 90% or 95% sequence identity with a sequence selected from sequences comprising SEQ ID NO:5, 11 or 12 and/or, wherein the C-terminal intein fragment has 100% sequence identity with a sequence selected from sequences comprising SEQ ID NO:6 or 13-18 or has at least 90% or 95% sequence identity with a sequence selected from sequences comprising SEQ ID NO:6 or 13-18.
Such isolated polypeptides provide novel split inteins ideally suited for protein modification and semi-synthesis due to their superior splicing yields and rates.
In various embodiments of this aspect of the invention the isolated polypeptide comprises at least one N-terminal intein fragment and at least one C-terminal intein fragment wherein the N-terminal intein fragment is selected from sequences comprising SEQ ID NO:5, 11 or 12 and the C-terminal intein fragment is selected from sequences comprising SEQ ID NO:6 or 13-18. Alternatively, the N-terminal intein fragment and the C-terminal intein fragment may be part of separate isolated polypeptides.
Besides their superior splicing yields and rates the novel split inteins are capable of efficient splicing at 37° C. This characteristic is advantageous as it renders the inteins more thermostable, better suited for expression of fusion proteins in organisms such as E. coli and in general broadens their practical utility for a range of applications.
In detail, a clear improvement over the wild-type AceL-TerL intein trans-splicing reactions at 37° C. could be observed for all the mutants. Rates were increased by about 2- to 14-fold (
Especially preferred combinations of N-terminal intein fragments selected from WTN, M1N, M3N and C-terminal intein fragments selected from sequences WTC, M1C, M2C, M3C, M4C, M5C, M6C are listed in Table 1.
As used herein the term “wild-type” refers to the phenotype of the typical form of a species as it occurs in nature. In relation to the provided intein sequences it should be noted that the term can also refer to artificially joined sequences, which themselves possess the wild-type succession of amino acids or nucleotides, respectively.
The term “splicing yield” as used herein refers to the amount of splice product produced. Ideally the intein mediated trans-splicing reaction would only result in splice product comprising the extein sequences attached to the intein fragments prior to the reaction. However, under unfavourable conditions or when using an intein, which is not suitable, the formation of cleavage side-product may occur. This lowers the overall splicing yield.
The term “splicing rate” as used herein refers to the change in concentration of the products per unit time. It is expressed using a rate constant k. The rate constant, k, which is temperature dependent, is a proportionality constant for a given reaction.
In still a further aspect the present invention relates to an isolated polypeptide wherein the polypeptide comprises the N-terminal intein fragment with SEQ ID NO:5 or 12 and the C-terminal intein fragment with SEQ ID NO:13. Alternatively, the N-terminal intein fragment and the C-terminal intein fragment may be part of separate isolated polypeptides.
Such an isolated polypeptide provides a novel split intein with even better splicing yields and rates.
In detail, these combinations spliced significantly faster with a beneficial ratio between splicing and cleavage yields (
In yet a further aspect the present invention relates to an isolated polypeptide as described above wherein the N-terminal intein fragment has 100% sequence identity with a sequence selected from sequences comprising SEQ ID NOs: 5, 28-33 or has at least 90% or 95% sequence identity with a sequence selected from sequences comprising SEQ ID NOs: 28-33 and/or, wherein the C-terminal intein fragment has 100% sequence identity with SEQ ID NO:27 or has at least 90% or 95% sequence identity with with SEQ ID NO:27. Alternatively, the N-terminal intein fragment and the C-terminal intein fragment may be part of separate isolated polypeptides.
As shown in
Moreover, it was unexpectedly found that the IntC with SEQ ID NO:27 is not only capable of splicing with the wild-type IntN sequence with SEQ ID NO:28, but is also capable of splicing with the IntN with SEQ ID NO:5. In addition, this cross splicing proceeds with both the original Cys1 of SEQ ID NO:5 and with a Seri at this position (cf.,
In addition, the fact that the IntC with SEQ ID NO:27 is capable of splicing with the IntN with SEQ IS NO 5, i.e. cross-splices, is advantageous as it substantially extends the possibilities for use of both intein fragments. In other words, it could for example be envisaged to pre-assemble—possibly in form of a kit—an IntC-EGFP fusion protein comprising SEQ ID NO:27 or merely the IntC fragment, e.g., for easy protein labelling. The ready IntC-EGFP fusion proteins could then be used with either the IntN of SEQ ID NO:5, SEQ ID NO:28-33, whichever one is available, cheapest to generate or for other reasons most suitable for easy and robust protein labelling.
In still a further aspect, the present invention relates to an isolated polypeptide further comprising at least one C-terminal extein or at least one N-terminal extein sequence.
The term “extein” or “extein sequence” as used herein refers to the peptide sequences that link to form a new polypeptide after the intein has excised itself during splicing. In other words, the N-terminal and C-terminal exteins (also termed ExtN and ExC) flank the IntN and IntC fragments, respectively prior to the trans-splicing reaction.
In various embodiments of this aspect of the present invention the isolated polypeptide comprises at least one C-terminal extein and least one N-terminal extein sequence.
In various embodiments of this aspect of the present invention the isolated polypeptide comprises exactly one C-terminal extein and exactly one N-terminal extein sequence.
In yet still other embodiments of this aspect of the present invention at least one of the peptide sequences of the isolated polypeptide selected from N-terminal intein, C-terminal intein, C-terminal extein and/or N-terminal extein is a recombinant protein.
In various embodiments, the extein sequence is heterologous with respect to the intein fragment to which it is coupled, i.e. the two sequences do not naturally occur together but have been artificially combined.
It is understood that all the above embodiments are similarly applicable to scenarios where the N-terminal intein fragment and the C-terminal intein fragment are part of separate molecules. In such cases each of the two molecules may comprise the respective extein sequence, i.e. the polypeptide comprising the N-terminal intein fragment comprises the N-terminal extein and the polypeptide comprising the C-terminal intein fragment comprises the C-terminal extein.
Through using extein sequences that are heterologous to the chosen intein sequences, the full power of split inteins can be exploited, as the extein sequences will be joined by a peptide bond following the trans-splicing reaction with virtually no trace of the previously existing intein sequences.
Thus, in a further aspect the invention relates to two isolated polypeptides as described above, for example in form of a combination or composition, wherein one of the isolated polypeptides comprises least one heterologous N-terminal extein fused to an N-terminal intein fragment having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100 sequence identity with the amino acid sequence set forth in any one of SEQ ID Nos. 2, 3, 4, 5, 28, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176 and 182, and wherein the second one of the isolated polypeptides comprises at least one C-terminal extein sequence fused to a C-terminal intein fragment having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100 sequence identity with the amino acid sequence set forth in any one of SEQ ID Nos. 6, 27, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 194, 195, 196.
All embodiments disclosed above in relation to one isolated polypeptide are similarly applicable to such embodiments where two isolated polypeptides are used. This particularly applies to the above described combinations of intein fragments and their combination with flanking sequences.
Advantageously, the complementary IntN and IntC fragments with their respective extein sequences are used together, as thereby the full potential of split inteins is realized, e.g. in applications such as modification of a protein, protein lipidation, protein immobilization, protein backbone semisynthetic, artificial control of protein splicing by light and use as a molecular switch. This is especially the case, if the extein sequences are heterologous to the intein fragments and possibly to each other. As mentioned above in such a case the extein sequences, which can be synthesized separately, will be joined by a peptide bond following the trans-splicing reaction with virtually no trace of the previously existing intein sequences.
In various embodiments of all aspect of the present invention the isolated polypeptide is a contiguous cis splicing polypeptide.
The term “cis splicing polypeptide” as used herein refers to a polypeptide, which has a continuous polypeptide sequence.
In a preferred embodiment the contiguous cis splicing polypeptide has the amino acid sequence of SEQ ID NO:7 or 8.
In other embodiments of all aspects of the present invention, the isolated polypeptide is a trans splicing polypeptide, i.e. has a discontinuous polypeptide sequence that needs complementation by another trans splicing polypeptide to become active. In such embodiments each of the trans splicing polypeptides as such as well as the combination of both, for example in form of a non-covalently associated complex, is intended to be encompassed by the present invention.
In a further aspect the present invention relates to an isolated polypeptide further comprising at least one component selected from a solubility factor, a marker, a linker, an epitope, an affinity tag, a fluorophore or a fluorescent protein, a toxic compound or protein and a small-molecule or a small-molecule binding protein.
As used herein the term “marker” refers to a component allowing detecting directly or indirectly the molecules having said marker. Thus, in some embodiments the marker is a label such as a fluorophore.
As used herein the term “linker” refers to a chemical moiety that connects parts of the isolated polypeptide or the nucleic acids described herein. Thus, a linker may be especially employed in the case of cis splicing polypeptides.
As used herein the term “epitope” refers to an immunogenic amino acid sequence.
As used herein the term “affinity tag” refers to a polypeptide sequence, which has affinity for a specific capture reagent and which can be separated from a pool of proteins and thus purified on the basis of its affinity for the capture reagent.
As used herein the term “fluorophore” means a compound or group that fluoresces when exposed to and excited by a light source, i.e. it re-emits light.
As used herein the term “fluorescent protein” refers to a protein that is fluorescent, e.g., it may exhibit low, medium or intense fluorescence upon exposure to electromagnetic radiation.
As used herein the term “small molecule” refers to any molecule, or chemical entity, with a molecular weight of less than about 1,000 Daltons.
These additional features are advantageous in order to increase the range of applications for which the isolated polypeptide can be used.
Preferred solubility factors are maltose binding protein (MPB), SUMO (small-ubiquitin like modifier), StreptagII, a His-tag, SBP (streptavidin binding peptide) and GST (glutathione-S-transferase.
Especially preferred is MBP as an N-terminal tag.
This has the advantage that protein expression and solubility are significantly increased.
Preferred markers are green and red fluorescent proteins (EGFP and mRFP), Gaussia princeps luciferase Gluc.
In various embodiments of this aspect of the present invention the isolated polypeptide comprises a linker fragment inserted between the at least two intein fragments.
In still other various embodiments of this aspect of the present invention the linker fragment is selected from the amino acid sequence MG or the amino acid sequence MGSGGSG.
This has the advantage that protein expression of the contiguous cis splicing polypeptide is improved.
In various embodiments of all aspect of the present invention the isolated polypeptide has the highest splicing yield and splicing rate at 8° C. or 37° C.
In a further aspect the present invention relates an isolated nucleic acid molecule comprising a nucleotide sequence encoding for at least one polypeptide described above or a homolog, mutant or complement thereof.
The term “variant” as used herein refers to a nucleic acid molecule which is substantially similar in structure and biological activity to a nucleic acid molecule according to one of the claimed sequences.
The term “mutant” refers to a nucleic acid molecule the sequence of which has one or more nucleotides added, deleted, substituted or otherwise chemically modified in comparison to a nucleic acid molecule according to one of the claimed sequences, provided always that the mutant retains substantially the same properties as the nucleic acid molecule according to one of the claimed sequences.
As used herein, the term “complement” refers to the complementary nucleic acid of the used/known/discussed nucleic acid. This is an important concept since in molecular biology, complementarity is a property of double-stranded nucleic acids such as DNA and RNA as well as DNA:RNA duplexes. Each strand is complementary to the other in that the base pairs between them are non-covalently connected via two or three hydrogen bonds. Since there is in principle—exceptions apply for thymine/uracil and the tRNA wobble conformation—only one complementary base for any of the bases found in nucleic acids, one can reconstruct a complementary strand for any single strand.
However, for double stranded DNA the term “complement” can also refer to the complementary DNA (cDNA). cDNA can be synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase.
In various embodiments of this aspect of the present invention the isolated nucleic acid molecule has or comprises SEQ ID NO:19-23.
In further embodiments of this aspect of the invention the isolated nucleic acid molecule is comprised in a vector, preferably a plasmid.
The term “vector”, as used herein, refers to a molecular vehicle used to transfer foreign genetic material into another cell. The vector itself is generally a DNA sequence that consists of an insert (sequence of interest) and a larger sequence. The purpose of a vector to transfer genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell.
The term “plasmid”, as used herein, refers to plasmid vectors, i.e. circular DNA sequences that are capable of autonomous replication within a suitable host due to an origin of replication (“ORI”).
Furthermore, a plasmid may comprise a selectable marker to indicate the success of the transformation or other procedures meant to introduce foreign DNA into a cell and a multiple cloning site which includes multiple restriction enzyme consensus sites to enable the insertion of an insert. Plasmid vectors called cloning or donor vectors are used to ease the cloning and to amplify a sequence of interest. Plasmid vectors called expression or acceptor vectors are specifically for the expression of a gene of interest in a defined target cell. Those plasmid vectors generally show an expression cassette, consisting of a promoter, the transgene and a terminator sequence. Expression plasmids can be shuttle plasmids containing elements that enable the propagation and selection in different host cells.
In further embodiments of this aspect of the at least one isolated nucleic acid molecule is comprised in a host cell.
The term “host cell”, as used herein refers to a transgenic cell, which is used as expression host. Said cell, or its progenitor, has thus been transfected with a suitable vector comprising the cDNA of the protein to be expressed.
In a further aspect the present invention relates to a protein expression system comprising an isolated polypeptide as described above or an isolated nucleic acid molecule as described above expressed from a plasmid in a host cell, e.g. E. coli.
In yet another aspect the present invention relates to a method using an isolated polypeptide as described above or an isolated nucleic acid molecule as described above, wherein the method is selected from the group consisting of modification of a protein, protein lipidation, protein immobilization, protein backbone semi-synthesis, regioselective protein side chain modification and artificial control of protein splicing by light, by a small molecule or a temperature change.
As used herein the term “protein lipidation” refers to the covalent modification of peptides, polypeptides and proteins with a variety of lipids, including fatty acids, isoprenoids, and cholesterol. Lipid modifications play important roles in the localization and function of proteins.
The term “semi-synthesis” as used herein refers to partial chemical synthesis, i.e. a type of chemical synthesis that uses compounds isolated from natural sources (e.g. plant material or bacterial or cell cultures) as starting materials. This is opposed to a total synthesis where large molecules are synthesized from a stepwise combination of small and cheap (usually petrochemical) building blocks.
The term “backbone semi-synthesis” as used herein refers to generation of a protein consisting of a polypeptide segment derived from recombinant protein expression and a segment obtain by organic peptide synthesis.
In a preferred embodiment of this aspect of the present invention the isolated polypeptide is modified in such a way it can be specifically induced to cleave, resulting in a separation of the intein and the N-terminal and/or C-terminal extein sequences. Such a modification can, for example, be achieved via point mutations in at least one of the extein sequences or by omitting one of the extein sequences.
Such a system could, e.g. be used for the purification of proteins with the subsequent cleavage of the employed affinity tag.
Protein trans-splicing (PTS) using inteins is especially well suited for these applications since the protein of interest (POI) will be assembled from two parts (fused as extein sequences to the intein fragments) and each of these fusion constructs can be prepared individually before the PTS reaction. Due to the small size the split intein fragments, one of the intein fusion proteins POI-IntN or IntC-POI can be obtained by using solid-phase peptide synthesis. Alternatively, both fusion proteins can be recombinant but treated individually with bioconjugation chemicals to regioselectively introduce a synthetic label.
Moreover, a method of protein modification employing an isolated polypeptide as described above can be carried out it in complex systems like a living cell or a cell extract.
In various embodiments of this aspect of the present invention the modification is a protein-terminal modification.
In various embodiments of this aspect of the present invention the N-terminal modification is protein labelling.
In a further aspect the present invention relates to a method for the ligation of at least one first peptide to at least one second peptide using an isolated polypeptide as described above or an isolated nucleic acid molecule as described above.
In this context it should be noted that the isolated polypeptide as described above or the isolated nucleic acid molecule as described above, respectively, can be used to generate one of the functional groups for the ligation reaction e.g. for native chemical ligation (NCL) or expressed protein ligation (EPL) reactions or can be employed to create the peptide bonds itself via intein mediated protein trans-splicing.
Thus, in various embodiments of this aspect of the present invention the method comprises covalently linking the N-terminus of the first protein to the C-terminus of the second protein, i.e. it is an intein mediated protein trans-splicing reaction.
In this context the isolated polypeptide described above is advantageous, because it allows for ligation at low concentrations and in the presence of other components.
In yet another aspect the present invention relates to the use of a polypeptide or a nucleic acid molecule as described above, wherein the use is selected from the group consisting of modification of a protein, protein lipidation, protein immobilization, protein backbone semisynthesis, regioselective protein side chain modification, and use as molecular switch. In various embodiments, the inteins and the intein-containing polypeptides described herein can be used for modification of proteins that have therapeutic utility, e.g. are used as pharmaceuticals. Such proteins include, without limitation, antibodies and antibody-like molecules.
In various aspects of this embodiment of the present invention the modification of a protein is selected from site-selective introduction of synthetic moieties into proteins and N-terminal modification.
In various aspects of this embodiment of the invention the described polypeptide or nucleic acids is used protein engineering or protein semi-synthesis.
Moreover, inteins have also been recognized as molecular switches that mediate protein splicing as an output signal only when a certain input signal is given. The latter represents the condition under which the intein is active. By fusing additional polypeptides that mediate a protein—protein interaction to the “free” termini of the split intein fragments the systems can be designed to either work as a biosensor or as an experimental tool to control biological activities that rely on the primary structure of the spliced polypeptide or protein.
In another aspect the present invention relates to a kit comprising a polypeptide or a nucleic acid molecule as described above.
In a preferred embodiment the kit comprises at least one polypeptide as described above comprising an IntN fragment as described above fused to a marker.
In various embodiments of this embodiment of the intention the kit further comprises at least one component selected from the group consisting of at least one vector, at least one resin, DTT, at least one plasmid, at least one expression host, at least one loading buffer, at least one antibody.
As used herein the term “expression host” refers to prokaryotic and eukaryotic expression system hosts, including but not limited to bacteria.
Such a kit is advantageous, inter alia, for ligation and labelling of recombinant proteins, as no proteases, which potentially splice the target protein, have to be used.
In one embodiment of this aspect of the present invention the kit comprises a vector encoding the IntC fragment, into which the protein of interest can be easily cloned. Thus, the IntC-protein of interest fusion protein can be easily expressed.
In addition, in one embodiment of this aspect of the invention the kit comprises the IntN fragment as synthetic peptide fused to a chemical modification desired for the protein of interest, e.g. a fluorophore, biotin etc. In a preferred embodiment the kit comprises the IntN fragment as synthetic peptide fused to a variety of chemical modifications.
In an alternative embodiment the IntC and IntN fragments along with their respective components are comprised in separate kits.
In various embodiments of all aspect of the present invention the splice site of the isolated polypeptide is at amino acids 24-25 from the intein's N-terminal position.
This is advantageous as due to the small size of the N-terminal intein fragment the POI-IntN complex can be obtained by using solid-phase peptide synthesis.
In various embodiments of all aspect of the present invention the N-terminal intein fragment of the isolated polypeptide comprises 24-25 amino acids and the C-terminal intein fragment of the isolated polypeptide comprises 104-105 amino acids.
This property has the advantage that small proteins and especially small intein fragments are more suitable for a range of applications such as chemical or biological, i.e. recombinant protein synthesis.
In various embodiments of all aspect of the present invention the isolated polypeptide is or is comprised in a recombinant protein and/or an antibody and/or a protein hormone.
Other embodiments are within the following non-limiting examples.
Several closely related phage genes with inteins were identified in metagenomic data from the Antarctic permanently stratified saline Ace Lake (F. M. Lauro, M. Z. DeMaere, S. Yau, M. V. Brown, C. Ng, D. Wilkins, M. J. Raftery, J. A. Gibson, C. Andrews-Pfannkoch, M. Lewis, J. M. Hoffman, T. Thomas, R. Cavicchioli, ISME J. 2011, 5, 879). The genes were of a T4 bacteriophage-type DNA packaging large subunit terminase, and were subsequently termed AceL-TerL inteins (from Ace lake terminase large subunit).
It was shown that these inteins have a novel split site corresponding to a probable surface loop region of the intein with no defined secondary structure following β-strand 3 and α-helix 1 of the typical intein structure (
The intein with SEQ ID NO:1, simply termed AceL-TerL intein hereafter, was chosen for functional characterization. To this end, the IntN of 25 aa was prepared by solid-phase peptide synthesis with three native N-extein residues, two lysine residues, and a 5(6)-carboxyfluoresceine moiety (FI-) to give pepWTN (FI-KKEFE-IntN). The IntC (aa 26-129) was recombinantly expressed in E. coli as a fusion with hexahistidine-tagged thioredoxin as a model protein (construct WTC-Trx-H6) and purified using Ni-NTA chromatography. Upon mixing of the two components spontaneous protein trans-splicing was observed (
As demonstrated in (
Although low temperatures like 8° C. appear ideal to preserve potentially fragile proteins of interest in in vitro experiments, expression of the intein fusion proteins in E. coli has to be performed at higher temperatures. Furthermore, inteins with higher thermostability should be beneficial for high activity in diverse sequence contexts, potentially also at lower temperatures, and for cellular applications.
Thus, in order to generate modified AceL-TerL inteins with a higher activity at 37° C., the AceL-TerL intein was converted into a contiguous, cis-splicing intein by fragment fusion (
Subsequently, a library encoding mutant inteins was created using error prone PCR (epPCR) and used to transform E. coli cells. Randomly picked kanamycin-resistant colonies that were selected on plates at 37° C. were then re-streaked on plates with kanamycin concentrations of up to 150 μg/ml. Plasmids isolated from resistant clones were analyzed by DNA-sequencing. Five different mutant inteins, termed M1 to M5 (Table 1), were selected and confirmed by Western blotting to have acquired splicing activity at this elevated temperature (
The effect of the mutations M1 to M6 was investigated. The IntC fragments of the M1 to M6 mutants, termed M1C to M6C, were expressed and purified as IntC-Trx-H6 fusion proteins, and the IntN parts of the M1 and M3 mutants, termed M1N and M3N, were included in two synthetic peptides of the format FI-KKEFE-IntN (pepM1N and pepM3N, respectively). A clear improvement over the wild-type intein protein trans-splicing reactions at 37° C. could be observed for all the mutants. Rates were increased by about 2- to 14-fold (
Moreover, the mutations seemed to have additive effects, as exemplified by the observation that the combined N46D and L55Q mutations (pepWTN+M1C) resulted in higher rates than the individual mutations (pepWTN+M5C and pepWTN+M6C, respectively) (
The IntN-mutation of the M3 mutant (Y3S) were combined with the IntC-mutations of the M1 (N46D, L55Q) or M2 (S38G, N461, N54D, L55Q) mutants (pepM3N+M1C and pepM3N+M2C). These combinations spliced ˜29-fold and ˜56-fold faster, respectively, than the wild-type at the selection temperature of 37° C. (
As the combinations of pepM3N+M1C and pepWTN+M1C surprisingly demonstrated a superior ratio between splicing and cleavage yields these were chosen for subsequent experiments. They were termed MX1 mutant (pepWTN+M1C) and M31 mutant (pepM3N+M1C) (Table 1 and
The maltose binding protein (MBP) was included as an N-terminal tag to give the construct MBP-IntC-TycB1 with the AceL-TerL MX1 and M31. MBP improved semi-synthetic protein trans-splicing of the wild-type AceL-TerL intein and mutants (data not shown). In particular, the MX1 mutant was efficiently expressed, well soluble, and spliced at 8° C. to give yields of 80-95% with a 13-fold higher rate than the M86 DnaB mutant intein and a 15-fold higher rate than the unevolved wild-type AceL-TerL intein (
In order to demonstrate the applicability of the AceL-TerL intein mutants for the chemical modification of a diverse range of proteins of interest, the MX1 mutant was fused with green and red fluorescent proteins EGFP and mRFP, Gaussia princeps luciferase Gluc, as well as the murine E2 conjugating enzyme Ubc9 and the human protease SENP1 from the SUMO pathway.
As shown in
The IntC encoding fragment of the VidaL_T4Lh-1 intein (nt: SEQ ID NO:121; aa: SEQ ID NO:117) was cloned into an expression vector coding for the construct SBP-(VidaL_T4Lh-1)C-Trx-His6 (SBP=streptavidin binding peptide, Trx=thioredoxin, His6=hexahistidine tag; amino acid (aa) sequence: SEQ ID NO:197; nucleotide (nt) sequence: SEQ ID NO:198). The protein was produced by overexpression in E. coli and purified from the supernatant after cell lysis using streptactin affinity chromatography. The IntN fragment of the intein (aa: SEQ ID NO:116) was synthesized by solid-phase peptide synthesis as a part of the peptide
Following mixing of both fragments (concentrations for IntN-construct 9 μM, for IntC-fragment 9 μM) in splice buffer (50 mM Tris/HCl, 300 mM NaCl, 1 mM EDTA, pH 7.0) with 2 mM TCEP (tris-carboxyethylphosphine) incubation was carried out at 25° C. Aliquots were removed at the indicated time points and quenched by mixing with SDS PAGE sample buffer and boiling at 95° C. for 5 min. Shown is an analysis of the samples on a Coomassie-stained SDS PAGE gel (see
The IntC encoding fragment of the VidaL_UvsX-2 intein (nt: SEQ ID NO:127; aa: SEQ ID NO:123) was cloned into an expression vector coding for the construct (VidaL_UvsX-2)C-Trx-His6 (Trx=thioredoxin, His6=hexahistidine tag; aa: SEQ ID NO:204; nt: SEQ ID NO:205. The protein was produced by overexpression in E. coli and purified using Ni-NTA-chromatography. The IntN fragment of the intein (SEQ ID NO:122) was synthesized by solid-phase peptide synthesis as a part of the peptide FI-ESGCLPKEAVVQIRLTKKGA-OH (SEQ ID NO:206; FI=5,6-Carboxyfluoresceine; IntN sequence underlined). Following mixing of both fragments (concentrations for IntN-construct 15 μM, for IntC-fragment 15 μM) in splice buffer (50 mM Tris/HCl, 300 mM NaCl, 1 mM EDTA, pH 7.0) with 2 mM TCEP (tris-carboxyethylphosphine) incubation was carried out at 25° C. Aliquots were removed at the indicated time points and quenched by mixing with SDS PAGE sample buffer and boiling at 95° C. for 5 min. Shown is an analysis of the samples on a Coomassie-stained SDS PAGE gel (see
In summary, novel inteins with an unusually short N-terminal fragment of only 15, 16 or 25 amino acids were identified and significantly improved mutants of these intein were generated. These intein fragments and the corresponding inteins respectively, can serve as powerful and generally applicable tools for the N-terminal chemical modification of proteins using semisynthetic protein trans-splicing. Advantages of this approach over chemical ligation reactions include the low required reactant concentrations, the absence of non-proteinogenic functional groups to facilitate the reaction, and the orthogonality to the cellular chemical environment. The high activity of the new split inteins at low temperatures like 8° C. is of particular advantage for in vitro labelling experiments with fragile proteins.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13196924.8 | Dec 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/077578 | 12/12/2014 | WO | 00 |
