Cloning And Expression Of Pre-Pro-Nattokinase Gene

Nattokinase,a kind of subtilisin,is a serine protease during atto fermenting by Bacillus subtilis which holds strong fibrinolytic activity.Experiments were carried out to clone pre-pro-nattokinase gene ,then to express it in Escherichia coli, and finaly to study the expression conditions.The chromosome DNA was isolated from Bacillus subtilis, Pre-pro-nattokinase gene was obtained by PCR assay and fused with cloning vector pUC19.The recombinant plasmid pNK was transformed into Escherichia coli DH5 α .According to sequence analysis showed , the obtained DNA was 1152bp and shared 100% homology with the reported sequence in GenBank. The obtained gene was fused with expression vector pBV220and pET30a.The recombined espression vector pBNK and pENK was then transformed into Escherichia coli HB101and BL21 (DE3) respectively. Temperature variation induced the transcription of the target gene in transformed bacterium pBNK6-HB101. The expression products were analyzed by SDS-PAGE. Comparition on the expression level of the target protein was made at different temperature,in different time and different medium. IPTG induced the...