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Applied and Environmental Microbiology, February 2005, p. 656-662, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.656-662.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli

Frontier Research Division, Fujirebio Inc., Hachioji, Tokyo, Japan¹

Received 29 April 2004/ Accepted 30 August 2004

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA₂₁₈) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 x 10⁴ E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA₂₁₈ fusion protein at 23 and 30°C but unexpectedly not at 37°C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA₂₁₈ was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA₂₁₈ fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.

Present address: School of Dentistry, Matsumoto Dental University, Shiojiri, Nagano 399-0781, Japan.

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