Cloning and expression of phospholipase $A_1$ gene from serratia sp. MK1 in E. coli = Serratia sp. MK1으로부터 Phospholipase $A_1$ 유전자의 Cloning 및 대장균에서의 발현

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Phospholipase $A_1$ gene from Serratia sp. MK1 was cloned in Escherichia coli DH5$\alpha$. 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 $37^\circ C$, 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.
Rhee, Joon-Shick이준식
한국과학기술원 : 생물과학과,
Issue Date
105855/325007 / 000943287

학위논문(석사) - 한국과학기술원 : 생물과학과, 1996.2, [ iv, 43 p. ]


Phospholipase $A_1$; Expression; Viable Cell Count; Plasmid Instability

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