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From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli . The expression vector was constructed so that the scFv gene (V L -linker-V H ) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15–20 mg per 1-liter flask cultivation.

A method convenient for isolation of DNA sequences capable of directing gene transcription in both organisms of E. coli and yeast is described. The method is composed of sequential steps of phenotypic selection for chloramphenicol resistance, first in E. coli and then in yeast. A series of promoter-probe, shuttle plasmid vectors between yeast and E. coli were constructed and utilized in the method.