Intermolecular protein splicing and its use in biotechnological applications

Inteins are selfish but harmless autocatalytic proteins that perform a post-translational modification, termed protein splicing. In protein splicing an intein excises itself off from the precursor protein and simultaneously ligates the flanking proteins together with a peptide bond. Inteins are found sporadically distributed in unicellular organisms, but their biological functions remain obscure. Importanly, inteins that are split into two can remain active and perform protein ligation by protein trans-splicing (PTS). In principle, PTS allows ligation of any two protein-sequences, with the only requirement being Ser, Thr, or Cys as the first residue downstream of the intein. This has inspired development of numerous biotechnological applications including protein semisynthesis, segmental isotopic labeling, and cyclization. Protein ligation by split inteins is, however, limited by the lengths, substrate specificity, orthogonality, and the reaction yields of the split inteins. The objective of this thesis was to advance the development of protein splicing as a protein-ligation tool. First, the split site of a natively split DnaE intein was shifted in order to engineer a split intein with shorter C-intein that could be easily chemically synthetized. The newly engineered split intein could perform protein ligation in high yields and was demonstrated to be in certain cases even better than the natively split intein. Encouraged by this, 21 more split inteins were engineered starting from four different inteins, guided by the three dimensional structures of these inteins. Split inteins were systematically tested for activity and orthogonality to evaluate their potential for biotechnological applications. Next, the scope was widened to bacterial intein-like (BIL) domains. BIL domains belong to the same superfamily with inteins but are distinct by their distribution and functions and have a wider variety of residues at the downstream junction. The first structure of a BIL domain was solved. It highlighted their homology to inteins as well as allowed engineering of split BIL domains. The split BIL domains could perform protein ligation also with Ala at the downstream splicing junction, although in minute yields, which could be the first step towards nucleophile-free protein ligation. Finally, discovery of a previously not reported intermolecular protein-splicing reaction, termed intein-mediated protein alternative splicing (iPAS), was described. Structural studies revealed that three-dimensional domain swapping is the underlying mechanisms of iPAS. iPAS makes it possible to increase diversity at protein level, without altering the genetic code, and could be used to control protein functions in concentration and expression-order dependent manner. Discovery of this new phenomenon could allow protein interference and is opening new insights into the possible biological functions of inteins.Proteiinien silmukointi työkaluna proteiinien muokkauksessa ja aktiivisuuden säätelyssä Tässä työssä on tutkittu proteiinien silmukointia sekä mahdollisuuksia hyödyntää sitä bioteknologian sovelluksissa. Työssä kehitettiin olemassa olevia proteiinien silmukointiin perustuvia ligaatio-menetelmiä sekä kuvataan uudenlainen intermolekulaarinen proteiinien silmukointi -mekanismi, joka mahdollistaa proteiinien aktiivisuuden säätelyn proteiinitasolla. Proteiinien silmukointi -domeenit voidaan jakaa kolmeen eri luokkaan, joista kahta, inteiinejä ja bakteerien inteiinin-kaltaisia (bacterial intein-like, BIL) domeeneja, on tutkittu tässä työssä. Inteiinit ovat yksisoluisista eliöistä löytyviä autokatalyyttisiä proteiini-domeeneja, jotka silmukoituvat irti esiproteiinista liittäen samalla vapautuvat päät yhteen peptidi-sidoksella. Inteiineistä ei tiedetä olevan hyötyä, muttei haittaakaan, isäntäorganismeilleen. Niitä voidaan kuitenkin hyödyntää useissa bioteknologian, biokemian ja biolääketieteen sovelluksissa, sillä ne mahdollistavat post-translationaalisen proteiinin muokkauksen. Suuri osa sovelluksista perustuu proteiinien trans-silmukointiin, joka on halkaistujen inteiinien välittämä proteiinien ligaatio -reaktio. BIL-domeenit voivat myös silmukoitua mutta niiden pääasiallinen tehtävä on katkaista viereinen proteiini irti. Päinvastoin kuin inteiinit, BIL-domeenien uskotaan olevan tärkeitä isäntäorganismeilleen mutta niille ei ole kehitetty bioteknologisia sovelluksia. Tässä työssä muokattiin uusia halkaistuja inteiinejä sekä tutkittiin näiden bioteknologisten sovellusten kannalta tärkeitä ominaisuuksia in vivo sekä in vitro. Työssä kuvataan uusia aktiivisia halkaistuja inteiinejä, joista muutamalla on sovellusten kannalta erityisen lupaavia ominaisuuksia. Lisäksi työ lisäsi tietämystä halkaistuja inteiinejä kehittäessä huomioitavista seikoista. Työssä ratkaistiin ensimmäinen BIL-domeenin kolmiulotteinen rakenne NMR-spektroskopian avulla. Tämä vahvisti näiden rakenteellisen homologian inteiinien kanssa sekä auttoi suunnittelemaan proteiinien ligaatioon soveltuvia halkaistuja BIL-domeeneja. Työssä osoitettiin, että halkaistujen BIL domeenien avulla on teoriassa mahdollista ratkaista inteiinien reaktiomekanismista juontuva rajoitus kohdeproteiineille, vaikka reaktion tehokkuutta tulisikin parantaa. Kolmantena osuutena työssä kuvataan inteiini-välitteinen vaihtoehtoinen proteiinien silmukointi (intein-mediated protein alternative splicing, iPAS). Rakennetutkimukset osoittivat reaktion johtuvan kolmiulotteisesta domeenien uudelleenjärjestäytymisestä. Ilmiö voitiin toistaa useilla eri inteiineillä sekä eri kohdeproteiineilla, mukaan lukien eräs luonnollisista isäntäproteiineista. Lisäksi iPAS:n osoitettiin mahdollistavan proteiinien aktiivisuuden häirinnän sekä pelastamisen. Näitä havaintoja voidaan hyödyntää uusien työkalujen kehittämisessä ja niillä voi olla merkitystä inteiinien biologisen merkityksen kannalta

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH)

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH)

Recombinant Spider Silk Protein and Delignified Wood Form a Strong Adhesive System

For developing novel fully biological materials, a central question is how we can utilize natural components in combination with biomimetic strategies in ways that both allow feasible processing and high performance. Within this development, adhesives play a central role. Here, we have combined two of nature's excellent materials, silk and cellulose, to function as an adhesive system. As an initial step in processing, wood was delignified. Without lignin, the essential microstructure and alignment of the wood remain, giving a strong scaffold that is versatile to process further. A recombinant spider silk protein was used as a fully biological and water-based adhesive. The adhesive strength was excellent with an average value of 6.7 MPa, with a maximum value of up to 10 MPa. Samples of different strengths showed characteristic features, with high tear-outs for weaker samples and only little tear-out for strong samples. As references, bovine serum albumin and starch were used. Based on the combined data, we propose an overall model for the system and highlight how multiple variables affect performance. Adhesives, in particular, biobased ones, must be developed to be compatible with the overall adherend system for suitable infiltration and so that their mechanical properties match the adherend. The engineering of proteins gives an unmatched potential for designing adhesive systems that additionally have desired properties such as being fully water-based, biologically produced, and renewable.Peer reviewe

Substrate specificities of inteins investigated by QuickDrop-cassette mutagenesis

Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the -1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.Peer reviewe

Molecular crowding facilitates assembly of spidroin-like proteins through phase separation

Gaining insights into the processes that transform dispersed biopolymers into well-ordered structures, such as soluble spidroin-proteins to spider silk threads, is essential for attempts to understand their biological function and to mimic their unique properties. One of these processes is liquid-liquid phase separation, which can act as an intermediate step for molecular assembly. We have shown that a self-coacervation step that occurs at a very high protein concentration (> 200 gl(-1)) is crucial for the fiber assembly of an engineered triblock silk-like molecule. In this study, we demonstrate that the addition of a crowding agent lowers the concentration at which coacervation occurs by almost two orders of magnitude. Coacervates induced by addition of a crowding agent are functional in terms of fiber formation, and the crowding agent appears to affect the process solely by increasing the effective concentration of the protein. Furthermore, induction at lower concentrations allows us to study the thermodynamics of the system, which provides insights into the coacervation mechanism. We suggest that this approach will be valuable for studies of biological coacervating systems in general.Peer reviewe

The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues