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KMID : 0578319960060030296
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Molecules and Cells
1996 Volume.6 No. 3 p.296 ~ p.302
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High-level Expression and Secretion of Serratia marcescens Metalloprotease Inhibitor in Bacillus subtilis by Aid of Subtilison Promotor and Signal Sequence
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Bae, Kwang Hee
Lee, Si Hyoung/Kim, Sun Taek/Lee, Sang Jun/Shin, Yong Chul/Byun, Si Myung
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Abstract
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Metalloprotease inhibitor (SmaPI) of a gram-negative bacterium, Serratia marcescens, was expressed and secreted in a heterologous host, Bacillus subtilis DB431, by aid of a subtilisin promotor and signal sequence. The DNA fragment containing the coding sequence for SmaPI was amplified by polymerase chain reaction, and the amplified-DNA product was inserted into the downstream region of a subtilisin signal sequence. The recombinant SmaPI expressed in Bacillus subtilis DB431 was secreted into the culture medium in a large amount. After cultivation for 32 h, the amount of SmaPI secreted into the culture medium reached about 100 mg/liter when estimated by measuring inhibitory activities toward Serratia marcescens metalloprotease (SMP). The NH₂-terminal amino acid sequencing analysis confirmed that authentic SmaPI and the recombinant SmaPI have the same NH₂-terminal amino acid sequences. The inhibitory activity of the purified recombinant SmaPI was found to be nearly equivalent to that of authentic SmaPI.
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