Semisynthesis of proteins using split inteins.

Abstract:

:Protein splicing is an autocatalytic reaction in which an internal protein domain, the intein, excises itself out of a precursor protein and concomitantly links the two flanking sequences, the exteins, with a native peptide bond. In split inteins, the intein domain is divided into two parts that undergo fragment association followed by protein splicing in trans. Thus, the extein sequences joined in the process originate from two separate molecules. The specificity and sequence promiscuity of split inteins make this approach a generally useful tool for the preparation of semisynthetic proteins. To this end, the recombinant part of the protein of interest is expressed as a fusion protein with one split intein fragment. The synthetic part is extended by the other, complementary fragment of the split intein. A recently introduced split intein, in which the N-terminal fragment consists of only 11 native amino acids, has greatly facilitated preparation of the synthetic part by solid-phase peptide synthesis. This intein enables the chemoenzymatic synthesis of N-terminally modified semisynthetic proteins. The reaction can be performed under native conditions and at protein and peptide concentrations in the low micromolar range. In contrast to chemical ligation procedures like native chemical ligation and expressed protein ligation, the incorporation of a thioester group and an aminoterminal cysteine into the two polypeptides to be linked is not necessary. We discuss properties of useful inteins, design rules for split inteins and intein insertion sites and we describe selected examples in detail.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Ludwig C,Schwarzer D,Zettler J,Garbe D,Janning P,Czeslik C,Mootz HD

doi

10.1016/S0076-6879(09)62004-8

subject

Has Abstract

pub_date

2009-01-01 00:00:00

pages

77-96

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(09)62004-8

journal_volume

462

pub_type

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