A phospholipase $A_1$ operon of Serratia sp.MK1 consists of plaA and plaB coding phospholipase $A_1$ and accessory protein respectively. Serratia.sp. MK1 secretes phospholipase $A_1$ into culture medium where there was two different sizes of the secreted phospholipase $A_1$. N-terminal signal sequence prediction and N-terminal amino acid sequencing of phospholipase $A_1$ showed that phospholipase $A_1$ is synthesized as a preproprotein which had a prepeptide as a signal sequence and a propeptide. The propeptide is supposed to be cleaved off by non-serine extracellular protease after secretion because serine protease inhibitor, PMSF,did not lower the propeptide processing. The size of mature phospholipase $A_1$ was 250 a.a while preproprotein of phospholipase $A_1$ had 320 a.a. We studied roles of propeptide and an accessory protein, PlaB on the restoring of phospholipase $A_1$ activity in the cell. The proprotein of phospholipase $A_1$ showed higher activity than mature phospholipase $A_1$ when expressed intracellularly in E.coli. plaB increased the extracellular phospholipase $A_1$ production when expressed in E.coli with phospholipase $A_1$ gene in two different plasmids. In addition, a refolding efficiency of denatured phospholipase $A_1$ was increased by the addition of PlaB. Accordingly,PlaB seemed to be a molecular chaperone which mediated protein folding and increased phospholipase $A_1$ activity. However, phospholipase $A_1$ activity was lowered in vitro when PlaB was added to the mature phospholipase $A_1$. Finally, we concluded that propeptide and PlaB were intramolecular chaperone which mediated the folding of phospholipase $A_1$ and additionally PlaB acted as an inhibitor. PlaB acted as a chaperone when phospholipase $A_1$ had a propeptide and an inhibitor when phospholipase $A_1$ was a mature protein.
Serratia sp.MK1에서 생산되는phospholipase $A_1$ 효소의 유전자는 phospholipase $A_1$을 지정하는 plaA gene과 accessory protein인 PlaB를 지정하는 plaB gene으로 구성되어 있다. Serratia sp.MK1에서 생산되는 phospholipase $A_1$은 cell 밖으로 분비되는데, culture media에서는 두 종류의 phospholipase $A_1$이 발견된다. N-terminal signal sequence prediction과 N-terminal amino acid sequencing을 한 결과 phospholipase $A_1$은 signal sequence와 propeptide를 가지는 preproprotein임을 알았다. Propeptide는 secretion후에 non-serine protease에 의해 잘려지는 것으로 추정된다. 왜냐하면, serine protease inhibitor인 PMSF를 처리해도 propeptide가 잘리는 것이 억제되지 않았기 때문이다. Preproprotein이 320개의 아미노산을 가지는 것에 비해 mature protein은 250개의 아미노산을 가진다. 우리는 propeptide와 accessory protein의 역할에 대해 연구했다. Proprotein형태로 발현되는 phospholipase $A_1$ 이 대장균에서 mature protein형태로 발현되는 phospholipase $A_1$에 비해 높은 활성을 보였다. 이것으로 propeptide가 intramolecular chaperone으로 작용함을 알았다. plaA gene과 서로 다른 vector에 plaB gene을 넣어 대장균에서 발현시키면, plaA gene만을 발현시킨 것보다 phospholipase $A_1$ 생산이 증가된다. 그리고, refolding 실험에서 PlaB를 첨가시키면 phospholipase $A_1$의 refolding 효과가 증가하였다. 따라서, PlaB는 단백질 folding을 돕고, phospholipase $A_1$ 활성을 증가 시키는 chaperone이다. 그러나, PlaB을 mature phospholipase $A_1$의 refolding buffer에 첨가하였을 때 활성이 감소하였고, plaB gene이 발현된 대장균의 lysate과 mature phospholipase $A_1$ solution을 혼합하였을 때 활성이 감소하였다. 결과적으로 PlaB는 phospholipase $A_1$의 folding을 도와주는 chaperone과 inhibitor로 작용할 수 있다. PlaB는 phospholipase $A_1$이 propeptide를 가지고 있을 때는 chaperone으로 작용하고, phospholipase $A_1$이 mature protein일 때는 inhibitor로 작용한다.
