RESUMO
An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.
Assuntos
Inteínas , Processamento de Proteína , Pyrococcus abyssi/enzimologia , Ribonucleotídeo Redutases/metabolismoRESUMO
The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 degrees Celsius or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 degrees Celsius or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.
Assuntos
Inteínas , Proteínas/genética , Proteínas/isolamento & purificação , Marcadores de Afinidade , Sequência de Bases , DNA Polimerase II/genética , DNA Polimerase II/isolamento & purificação , DNA Arqueal/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Técnicas In Vitro , Plasmídeos/genética , Processamento de Proteína , Pyrococcus abyssi/enzimologia , Pyrococcus abyssi/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Resinas Sintéticas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C terminus, is 9.3 x 10(-6) s(-1) at 60 degrees C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 x 10(-4) s(-1). The first order rate constant of C-terminal cleavage is 1.2 x 10(-5) s(-1) with Gln at the C-terminal position, 2.8 x 10(-4) s(-1) with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.
