Liming Ge

Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: influence of thioredoxin reductase (TrxB)

The cytoplasmic expression of a functional antibody (Ab) fragment, containing the correct intradomain disulfide bonds, was investigated in E. coli. We used a single-chain Fv (scFv) fragment of the levan-binding Ab ABPC48, which was shown to be functional only in the presence of the disulfide bonds. Significant amounts of functional, disulfide-containing scFv could be produced in the cytoplasm of E. coli in the absence of thioredoxin reductase (TrxB) activity. The amount of soluble protein remained largely unchanged by this null mutation. A stronger promoter did not result in further improved yields of functional Ab fragment, despite much higher protein production, suggesting that inefficient disulfide formation was still limiting the yield of active scFv. This method of expressing functional Ab fragments in the cytoplasm of E. coli may be important for screening and selection systems.

Functional genomics with protein-protein interactions.

Knowing the sequence of a gene does not mean knowing its function. Although, information stored at the DNA level can be used to predict biological processes, proteins are the final executors of the various response programs of a cell. Transient information, like posttranslational modifications or interactions among proteins, cannot be deduced from DNA sequences. The rapid accumulation of large amounts of DNA sequence data in genomics projects has led to an increasing demand for powerful tools to analyze proteins and their behaviour at a large scale. This review aims to compare different technologies used for identification of interacting proteins and discusses recent developments in the field of high-throughput protein-protein interaction mapping.