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Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system

Paper ID Volume ID Publish Year Pages File Format Full-Text
23313 43431 2013 12 PDF Available
Title
Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system
Abstract

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.

► Functional scFv molecules are produced in eukaryotic in vitro translation systems. ► The systems contain vesicles derived from the endoplasmic reticulum of insect cells. ► ScFv molecules are co-translationally translocated into the lumen of these vesicles. ► ScFv molecules are released from insect vesicles by detergent containing buffer. ► Released antibody fragments are well-suited for label-free binding studies.

Keywords
CLSM, confocal laser scanning microscopy; C-SII, strep-tag antisense adapter primer; DDM, n-Dodecyl-β-maltoside; DTT, Dithiothreitol; E. coli, Escherichia coli; E-PCR, Expression-PCR; ER, endoplasmic reticulum; EYFP, enhanced yellow fluorescent protein; F
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Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 2, March 2013, Pages 220–231
Authors
, , , , , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us