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-ac...

Full description

Saved in:
Bibliographic Details
Published in:Current microbiology 2007-09, Vol.55 (3), p.228-233
Main Authors: Tao, Ke, Yu, Xiaoqi, Liu, Yun, Shi, Guanying, Liu, Shigui, Hou, Taiping
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Bacteria
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Bacterial Proteins - metabolism
Base Sequence
Cloning
E coli
Enzymes
Escherichia coli
Grasshoppers - microbiology
Microbiology
Molecular Sequence Data
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Resins
Serratia marcescens
Serratia marcescens - genetics
Serratia marcescens - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0343-8651
1432-0991
DOI:10.1007/s00284-007-0096-z