Cloning, expression, and purification of insecticidal protein Pr596 from locust pathogen Serratia marcescens HR-3.
A novel insecticidal protein (Pr596) produced by Serratia marcescens HR-3 was found be a metalloprotease and responsible for insecticidal activity toward locusts. Two pairs of primers were designed to amplify Pr596, a putative open reading frame (ORF) by similarity search and the N-terminal amino-acid sequence of insecticidal protein. The results revealed that the ORF consisted of 1464 nucleotides encoding a protein of 487 amino-acid residues. Pr596 was cloned into expression vector pET32a(+) and was expressed in Escherichia coli BL21 (DE3)/pLysS strain with isopropyl-beta-D-thiogalactopyranoside induction. The Pr596 was found to be highly expressed as inclusion bodies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pr596 inclusion bodies were isolated and subjected to Ni-NTA His Bind Resins (Pharmacia, Germany). Pr596 purified and refolded was revealed by SDS-PAGE and had proteolytic activity and insecticidal activity. Results suggested that there is a potential to develop this protein to be used as an alternative locus control agent.
Tao
Ke
K
Key Laboratory of Bio-resource and Eco-environment, Ministry of Education, Sichuan University, Chengdu 610064, PRC.
Yu
Xiaoqi
X
Liu
Yun
Y
Shi
Guanying
G
Liu
Shigui
S
Hou
Taiping
T
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D006110:Grasshoppers / Q000382:microbiology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012706:Serratia marcescens / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins