Abstract
In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library.
Highlights
In vitro selection by display methods has been an effective tool in the field of protein engineering and especially has been used to engineer recombinant antibodies for various biological applications [1]
Because the Fab is more native-like than the single-chain Fv, which is the other commonly used recombinant antibody format for in vitro selection, the Fab fragment format makes it able to select more practical antibodies [3]
Cell-free translation-based methods such as ribosome display [4] and mRNA display [5] are being used for in vitro selection of antibodies due to its advantage of permitting speedier selection from larger size libraries than cell-based methods. These cell-free translation-based methods are limited to select single-chain Fv (scFv) due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome
Summary
In vitro selection by display methods has been an effective tool in the field of protein engineering and especially has been used to engineer recombinant antibodies for various biological applications [1]. Cell-free translation-based methods such as ribosome display [4] and mRNA display [5] are being used for in vitro selection of antibodies due to its advantage of permitting speedier selection from larger size libraries than cell-based methods. These cell-free translation-based methods are limited to select scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Since mRNA display is capable of selecting candidates from a more diverse library and designing a more
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