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Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library.

Abstract：

:This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.

journal_name

Nat Biotechnol

journal_title

Nature biotechnology

authors

Cesaro-Tadic S,Lagos D,Honegger A,Rickard JH,Partridge LJ,Blackburn GM,Plückthun A

doi

10.1038/nbt828

subject

Has Abstract

pub_date

2003-06-01 00:00:00

pages

679-85

issue

6

eissn

1087-0156

issn

1546-1696

pii

nbt828

journal_volume

21

pub_type

杂志文章

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