CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, , was cloned and expressed in a heterologous host (Jeong ., 2007). In this study, the promoter of was replaced with other stronger promoters (P, P, P, P) of spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into WB600. The P10 promoter conferred the highest fibrinolytic activity, , 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest expression was observed when two copies of the P promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.
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| http://dx.doi.org/10.4014/jmb.2103.03027 | DOI Listing |
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