Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes.

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase.

An improved protein bioreactor: efficient product isolation during in vitro protein biosynthesis via affinity tag.

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity.

Cell-free expression of two single-chain monoclonal antibodies against lysozyme: effect of domain arrangement on the expression.

Single-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli [Tsumoto et al. (1995) Biochem.

Ribonucleolytic activities in the Escherichia coli in vitro translation system and in its separate components.

mRNA stability is a limiting parameter for the efficiency of in vitro protein biosynthesis. In order to develop strategies to prolong the mRNA half-life, we investigated the ribonuclease activities in the complete Escherichia coli system, in the separate cell fractions 70S ribosomes and S-100 and in the non-cellular fraction. Our results imply that the amount of ribonucleolytic activities and the insensitivity to placental RNase inhibitor in the complete system are due to the 70S ribosome fraction, whereas the generation of small degradation products is due to the S-100 fraction.

The potentials of the in vitro protein biosynthesis system.

Recent developments indicate that with the in vitro protein biosynthesis system a new technology emerges, which will find in the future its application in biotechnology. Up to date the best system described produces 2 mg of proteins per 0.5 ml after 100 h.