In the present study, we investigated a microplate-based culture (MBC) of Escherichia coil for the high-throughput, high-level production of PS-tag-fused scFvs (scFv-PS) in insoluble form. The Overnight Express (TM) Autoinduction System (OE system) was adopted to skip the laborious induction step of the addition of IPTG. ScFv and scFv-PS began to be expressed after 6 h by conventional flask culture (FLC) and by MBC when utilizing the OE system, and similar specific productivity levels were attained during cultivation. In MBC, an important factor that directly affected the production levels was rotational speed during cultivation, suggesting that the mass transfer of oxygen was rate-limiting. In a comparison of the productivity of flask cultures utilizing the 2YT-IPTG and OE systems, MBC utilizing the OE system was the highest, with approximately 1 mg of insoluble scFv-PS obtained from each well under optimal conditions (1400 rpm). The results of SDS-PAGE and a cross-contamination check indicated that very similar cultivation conditions were attained in each well, without cross-contamination. Thus, MBC using the OE system is very useful for the high-throughput, high-level production of scFv-PS, which can be activated on the surface of hydrophilic PS plates by solid-phase refolding. Therefore, the production of a variety of specific scFv-PSs for cytokines and biomarkers will make possible the construction of sensitive and low-cost antibody microarrays, which will be very useful in clinical diagnosis and biochemical research. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.