토양 등의 인 제한환경에서 특이적으로 발현하는 벡터를 구축하기 위해서 Enterobacter areogenes의 phoA 유전자의 promoter가 든 pEAAP를 구축하였다. pEAAP는 pET-22b(+)을 BglII와 XbaI으로 절단하여 T7 promoter와 lac operator를 제거하고pho box가 포함된 phoA promoter를 삽입하여 구축하였다. pEAAP가 인 제한 환경에서 특이적으로 발현되는지 조사하고자 Bacillus subtillis var. amyloliquefaciens (KCTC 8913P)의 Phytase유전자인 Bsa-phy1을 도입한 pEAPHY1을 구축하였다. CK-PHY1 (pEAPHY1을 도입한Escherichia coli JM109)는 인 제한 환경에서 41 kD)의 Bsa-Phy1을 발현하였다. 또한, CK-PHY1은 phytate를 유일한 인산원으로 첨가된 고체배지에서 phytate를 분해하여 투명대를 형성하였다.
To induce recombinant protein under phosphate restricted conditions such as soil, we have constructed the expression vector (pEAAP) with phoA gene promoter of Enterobacter aerogenes. To construct the pEAAP, deletion of the T7 promoter and lac operator from pET-22b(+) by BglII-XhoI digestion and addition of the phoA gene promoter (containing the pho box) were performed. To test pEAAP as an expression vector controled by phosphate limitation, pEAPHY1 was constructed with the phytate gene (Bsa-phy1) of Bacillus subtillis var. amyloliquefaciens (KCTC 8913P). Under the phosphate-limitation condition, CK-PHY1 ( Escherichia coli JM109 was transformed with pEAPHY1) expressed the 41 kD Bsa-Phy1 . Also CK-PHY1 formed the clear zone in solid medium containing phytate as a sole phosphate source.
