Shaorong Chong

SINGLE-COLUMN PURIFICATION OF FREE RECOMBINANT PROTEINS USING A SELF-CLEAVABLE AFFINITY TAG DERIVED FROM A PROTEIN SPLICING ELEMENT

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.

Modulation of Protein Splicing of the Saccharomyces cerevisiae Vacuolar Membrane ATPase Intein

Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (−1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the −1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of −1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.

Protein Splicing of the Saccharomyces cerevisiae VMA Intein without the Endonuclease Motifs

The protein splicing element (intein) of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiaecatalyzes both protein splicing and site-specific DNA cleavage. It has been demonstrated that the conserved splice junction residues are directly involved in protein splicing and the central dodecapeptide motifs are required for DNA cleavage. To examine whether the splicing activity of the intein can be structurally separated from the endonuclease motifs, we made large in-frame deletions at the central region of the intein. We demonstrate for the first time that protein splicing can proceed efficiently after the removal of the central region of the intein including the endonuclease motifs. Our results suggest that the N- and C-terminal regions of the Sce VMA intein may form a separate domain that is not only catalytically sufficient for protein splicing but also structurally independent from the endonuclease domain.

[24] Fusions to self-splicing inteins for protein purification

Publisher Summary This chapter describes the way inteins can be used to affect the self-catalyzed cleavage of fusion proteins at highly specific sites. To make use of the self-cleaving activity of inteins as tools for protein purification, it is necessary to prevent protein splicing by strategic amino acid replacements at either the upstream or the downstream splice junction. Depending on the type of amino acid substitution used, protein cleavage can be induced at either the upstream or the downstream splice junction. Different inteins vary in the efficiencies with which N- or C-terminal cleavage can be induced and in the compatibility of these cleavage reactions with the extein residues adjacent to the cleavage site. The decision of whether to fuse the C or the N terminus of the target protein to the intein also has important implications on the efficiency of protein expression and purification yield. The expression of heterologous proteins in Escherichia coli sometimes results in the formation of inclusion bodies or is inefficient because of the incompatibility of codon usage in the foreign gene with E. coli coding preferences. Both of these problems are most pronounced when the foreign polypeptide constitutes the N-terminal segment of the fusion protein and can often be reduced by fusing the target protein to the C terminus of the intein.

Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence. The intein-GFP system may provide a simple route for monitoring real time soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.

Intein-mediated Protein Purification

Protein-splicing elements are remarkable multifunctional protein domains that mediate their post-translational excision from a precursor protein. The intervening sequence (called the intein) plus the first carboxy-terminal amino acid of the surrounding host protein (called the extein), direct cleavage of the peptide bonds on both sides of the intein and the formation of a native peptide bond between the two extein fragments. The majority of inteins also encode a homing endonuclease subdomain that initiates mobilization of the intein gene into an extein gene homolog that does not contain the intein M. Understanding the mechanism of protein splicing has led to a variety of intein-mediated applications. This chapter describes the use of modified inteins in protein purification and the special chemical reactivities of proteins isolated after thiol cleavage when the carboxy-terminus of the target protein is fused to an intein vector.