A genetic system yields self-cleaving inteins for bioseparations

Abstract

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Gelatinase-mediated migration and invasion of cancer cells

“…Furthermore, minimal inteins with full cleavage activity have only 130-160 amino acids (Derbyshire et al, 1997;Mathys et al, 1999;Wood et al, 1999). With a minimal sized fusion partner, the protein expressing cells do not have to consume their metabolic reserves for expressing large unwanted proteins thus maximising the peptide yield.…”

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

“…The question of whether the chemical steps are catalyzed by a single or two separate active sites is a matter of debate; however, the catalytic sites responsible for Nand C-terminal cleavage steps in the Ssp DnaB intein do not appear to share common components and can proceed independently. The C-terminal cleavage reaction of the Ssp DnaB mini-intein, the Pyrococcus species B-D DNA polymerase intein, and the Mycobacterium tuberculosis RecA intein appear not to depend on the first two steps in the splicing pathway (12,19,45). On the other hand, because of the close proximity of the two junctions or active sites and the intricate balance of the three chemical steps, certain mutations introduced to one junction have been shown to affect the cleavage reaction at the other junction (14,20).…”

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

“…To this end, rapid methods are desired to screen for compounds capable of blocking protein splicing. Three in vivo methods for detection of splicing and nonsplicing intein variants were recently published (1,3,11).This report describes a new genetic selection system for the identification of splicing and nonsplicing intein variants inserted into phage RB69 DNA polymerase. The method is based on growth versus lysis of Escherichia coli cells infected with conditionally defective T4 gp43 Ϫ phage, which contains amber mutations in the T4 DNA polymerase gene (gene 43) that render the phage inviable in nonsuppressor strains.…”

“…The replica plating method for primary screening will enable screening of large numbers of clones in experiments aimed at isolating expressed peptides that inhibit protein splicing or for exploring residues involved in intein structure and function as described elsewhere (11). It is anticipated that compounds capable of inhibiting protein splicing in this context will function also as inhibitors of inteins in their natural insertion sites.…”

“…To this end, rapid methods are desired to screen for compounds capable of blocking protein splicing. Three in vivo methods for detection of splicing and nonsplicing intein variants were recently published (1,3,11).…”