||Cytoplasmic expression of proteins in E. coli encounters problems like protease degradation. While secretion of proteins to the periplasm partly solves the problems, excretory production may provide better solutions. In our laboratory, the Tac and the LacUV5 excretion cassettes have been employed for the studies of protein excretion in E. coli. Using these cassettes, several proteins have been successfully excreted. Since excretion of larger proteins appears to be more lethal than smaller ones to the host, the effect of molecular size on protein excretion was investigated. One to three copies of the hEGF gene were precisely fused to the ompA leader sequence in the LacUV5 cassette. E. coli JM101 transformants containing the construct with one copy of the hEGF gene survived better and excreted a higher level of the recombinant product than did the ones with two or three copies in time-course studies. To investigate whether the cytoplasmic protein β-galactosidase might be excreted using the LacUV5 cassette, the coding sequences for the full-length as well as the α-peptide (60 a. a.) of β-galactosidase were fused to the ompA leader sequence in the cassette. Transformants harboring recombinant constructs formed by either the full-length lacZ gene or the lacZ' gene did not survive well. Plasmid analysis indicated that the coding sequence for the OmpA signal peptide was scrambled, suggesting that the E. coli host might not be able to excrete cytoplasmic proteins even in the presence of a signal peptide. To investigate the effect of the α-peptide of β-galactosidase (60 a. a.) on hEGF excretion, two hybrid constructs encoding foreign products with 53 a. a. between hEGF and the α-peptide were cloned in the LacUV5 cassette downstream from the ompA leader sequence. Despite an expectancy of the two gene fusions to yield hybrid proteins sharing similar molecular sizes, DNA sequencing revealed that only the hEGF-lacZα fusion was accurately maintained in E. coli. Moreover, E. coli transformants harboring the hEGF-lacZα fusion construct were found to have high levels of plasmid stability and cell viability, indicating that the relative position between hEGF and the α-peptide in a hybrid protein could critically affect the success of protein expression / excretion by E. coli.