1997
DOI: 10.1038/nbt0197-79
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Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones

Abstract: To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence o… Show more

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Cited by 176 publications
(130 citation statements)
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“…S1), and have yields of scFvs and Fabs that are ~10-to 1,000-fold higher than in other E. coli-based high-throughput screening (HTS) expression systems. 14,21,25,26 Antibodies produced in this format are easily purified and tested using functional in vitro and cellbased assays. For example, the cell-free produced anti-hIL-23 scFv bound specifically to the p19 subunit of human IL-23, 27 with a K D of 10 nM, as measured by surface plasmon resonance (SPR; Fig.…”
Section: Resultsmentioning
confidence: 99%
“…S1), and have yields of scFvs and Fabs that are ~10-to 1,000-fold higher than in other E. coli-based high-throughput screening (HTS) expression systems. 14,21,25,26 Antibodies produced in this format are easily purified and tested using functional in vitro and cellbased assays. For example, the cell-free produced anti-hIL-23 scFv bound specifically to the p19 subunit of human IL-23, 27 with a K D of 10 nM, as measured by surface plasmon resonance (SPR; Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Improvements in folding of heterologous proteins in the presence of chaperones and/or folding catalysts have been achieved in cell-free translation (Ryabova et al, 1997) as well as in E. coli (Georgiou and Valax, 1996;Wall and Pluckthun, 1995). However, some of these proteins need to be expressed in stoichiometric amounts and co-expression of additional proteins complicates production with regard to plasmid stability and induction and therefore the potential to produce the target protein is decreased.…”
Section: Introductionmentioning
confidence: 99%
“…We then carried out the transcription and translation steps separately, the latter being performed using a rabbit reticulocyte system. By uncoupling transcription and translation, translation was possible without a reducing agent, potentially resulting in greater functional expression of single-chain antibodies (Ryabova et al, 1997). In addition, the rabbit reticulocyte system has lower RNase activity than an E. coli ribosome display system, making the selection conditions less complex (Hanes et al, 1999).…”
Section: Discussionmentioning
confidence: 99%