Kenneth V. Mills

Protein Splicing in Vitro with a Semisynthetic Two-component Minimal Intein

Protein splicing elements, or inteins, catalyze their own excision from flanking polypeptide sequences, or exteins, thereby leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. A trans-splicing system, using separately purified and expressed N- and C-terminal intein fragments of about 100 amino acids each, fused to appropriate exteins, was recently derived from the Mycobacterium tuberculosis RecA intein (Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3543–3548). We have replaced the C-terminal intein fragment of this system with synthetic peptides comprising 35–50 of the C-terminal residues of the RecA intein. The N-terminal intein fragment and the synthetic peptide were reconstituted by renaturation from guanidinium chloride. In the absence of added reductants, a disulfide-linked dimer of the N-terminal fragment and the peptide accumulated and could be induced to splice by reduction of its disulfide bond. The intermediate and spliced products were identified by polyacrylamide gel electrophoresis, mass spectrometry, and derivatization with thiol-reactive biotin followed by Western blotting with a streptavidin-enzyme conjugate. This is the first example of protein splicing involving a synthetic intein fragment and opens the way for studying the active site structure and function of the intein by the use of different synthetic peptides, including ones with non-natural amino acids.

Recent advances in in vivo applications of intein-mediated protein splicing

Intein-mediated protein splicing has become an essential tool in modern biotechnology. Fundamental progress in the structure and catalytic strategies of cis- and trans- splicing inteins has led to the development of modified inteins that promote efficient protein purification, ligation, modification and cyclization. Recent work has extended these in vitro applications to the cell or to whole organisms. We review recent advances in intein-mediated protein expression and modification, post-translational processing and labeling, protein regulation by conditional protein splicing, biosensors, and expression of trans-genes.

Protein Splicing: How Inteins Escape from Precursor Proteins

Inteins are natures escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled.

Characteristics of protein splicing in trans mediated by a semisynthetic split intein

Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans-splicing reaction mediated by a naturally expressed, approximately 100-residue N-terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C-terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37 degrees C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C-terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N-terminal fragment and either the naturally expressed 107-residue C-terminal portion of the intein, much smaller synthetic peptides, or the 107-residue C-terminal intein fragment modified by fusion of a maltose binding protein domain to its N-terminus. The ability to use different types of the C-terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation.

The mechanism of intein-mediated protein splicing: variations on a theme.

Intein-mediated protein splicing is facilitated by four separate but coordinated nucleophilic displacement reactions that result in the excision of the intein and the ligation of the flanking polypeptides, called the exteins. These reactions are catalyzed by the intein plus the first downstream extein amino acid without the assistance of cofactors or auxiliary enzymes. Non-canonical inteins missing conserved nucleophilic residues at the N- or C-terminus likely splice using variations of the standard mechanism.

Intramolecular Disulfide Bond between Catalytic Cysteines in an Intein Precursor

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.

Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi

Background: The PolII intein from the hyperthermophilic Pyrococcus abysii only splices at very high temperature. Results: NMR structure, dynamics, and mutagenesis of Pab PolII intein have been characterized. Conclusion: The Pab PolII intein has unique structural and dynamic features that may contribute to its higher temperature for optimal activity. Significance: Pab PolII intein is an ideal candidate for protein engineering. Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 Å and a heavy atom root mean square deviation of 0.93 Å. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra β-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.

Internal Disulfide Bond Acts as a Switch for Intein Activity

Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA polymerase II from Methanoculleus marisnigri (Mma) can promote protein splicing. However, protein splicing can be prevented or reduced by overexpression under nonreducing conditions because of the formation of a disulfide bond between two internal intein Cys residues. This redox sensitivity leads to differential activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control of in vivo protein splicing in an intein derived from an anaerobe that can occupy multiple environments hints at a possible physiological role for protein splicing.

Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum

An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.

Biochemical Mechanisms of Intein-Mediated Protein Splicing

This chapter discusses the mechanism of the self-catalyzed process by which inteins promote both their own excision from a host protein and the direct linkage of the flanking host protein segments, the N- and C-exteins, by a peptide bond. The majority of inteins have a nucleophilic amino acid at their N-terminus and asparagine at their C-terminus and are linked to a C-extein with an N-terminal nucleophilic amino acid. These canonical inteins promote protein splicing by a four-step mechanism of sequential acyl rearrangements. Non-canonical inteins, which lack either the N-terminal nucleophile or the C-terminal asparagine, promote protein splicing by a variant of this mechanism or promote protein cleavage rather than splicing. A remarkable feature of the protein splicing process is that it involves multiple steps that are chemically autonomous yet proceed in a highly coordinated manner without side reactions unless perturbed by mutation, unnatural exteins, or non-physiological conditions. The factors that may serve to integrate protein splicing into a system that ordinarily operates efficiently without side reactions are discussed.