Phospholipase $A_1$ gene from Serratia sp. MK1 was cloned in Escherichia coli DH5α. The presence of two open reading frames of 966 and 705 nucleotides was found from nucleotide sequences of cloned gene. Two open reading frames were designated as phlA and phlB, respectively. First open reading frame, phlA, encoded a 33.4 kDa polypeptide of 321 amino acid residues, which was designated PLA. The start codon of second open reading frame was 5 bases upstream of stop codon of phlA. Second open reading frame, phlB, encoded a 25.7 kDa polypeptide of 234 amino acid residues, which was designated PLB. The gene structure and deduced amino acid sequences revealed high homology to those of Serratia liquefaciens.
When E.coli containing pTrAB was cultured at 37℃, phospholipase $A_1$ was formed about 20% of total proteins for 4 hours after induction at final 0.8mM IPTG. Moreover, phospholipase $A_1$ activity from supernatant was also detected on selective agar plates. It was phlA gene product (PLA) that had phospholipase $A_1$ activity.
Expressed phlA gene product was detected in both cells and supernatant, as a result of secretion through a mechanism in which was involved by phlB gene product, or cell lysis by phospholipase $A_1$ activity degrading cell membrane. Though a portion of this phospholipase $A_1$ was secreted or leaked into the culture medium from E.coli host cells, the rest of overexpressed phospholipase $A_1$ were intracellular as enzymatically inactive and insoluble protein. Overexpression of cloned phospholipase $A_1$ is toxic to the host and may be resulted in problems of cell lysis and expression plasmid instability.
Serratia sp. MK1 으로부터의 포스포리파제 $A_1$ (phospholipase $A_1$) 유전자를 Escherichia coli DH5α에서 cloning하고, 염기 서열을 분석하였다. 2개의 open reading frame이 존재하였고 각각 phlA와 phlB로 명명하였으며 966개 705개의 염기쌍으로 구성되어 있었다.
첫 번째 open reading frame(phlA)은 321개의 아미노산으로 구성되어 33.4 kDa의 포스포리파제 활성을 나타낸 단백질(PLA)을 code 하였다. 두 번째 open reading frame(phlB)은 234개의 아미노산으로 구성된 25.7 kDa의 단백질(PLB)을 code하였다. 각각의 단백질은 Serratia liquefaciens에 대하여 72%와 71%의 동일성을 나타내었다.
phlA와 phlB를 모두 포함하는 E.coli JM109은 최종적으로 0.8mM의 IPTG 농도하에서 4시간 후에 가장 높은 발현 양을 나타내고, 전체 세포 단백질의 약 20%를 차지하였다. 포스포리파제 활성은 또한 세포 배양 상등액에서도 나타나며 이것은 PLA일부가 phlB gene product가 ？된 경로로 분비되거나, 세포막을 손상시키는 포스포리파제 활성에 의한 cell lysis의 결과이다. 반면에 발현된 나머지 단백질은 세포 내부에 활성 없는 형태로 축적되었다.
포스포리파제 $A_1$의 발현은 E.coli 세포에 toxic하여 cell lysis를 유발하였고, 발현 plasmid의 instability의 문제가 야기되었다.
