Paper Information

Journal:   IRANIAN JOURNAL OF PUBLIC HEALTH   AUGUST 2014 , Volume 43 , Number SUPPLEMENT 2; Page(s) 26 To 26.
 
Paper: 

RECOMBINANT NLP PRODUCTION FROM STREPTOMYCES CYANEOFUSCATUS UTMC 2101IN E. COLI

 
 
Author(s):  PAPIRAN REYHANEH*, HAMEDI JAVAD, MOGHIMI HAMID
 
* DEPARTMENT OF MICROBIAL BIOTECHNOLOGY, SCHOOL OF BIOLOGY AND CENTER OF EXCELLENCE IN PHYLOGENY OF LIVING ORGANISMS, COLLEGE OF SCIENCE, UNIVERSITY OF TEHRAN, TEHRAN, IRAN
 
Abstract: 

Background: Necrosis and ethylene inducing like family of proteins (NLPs) are microbial-derived herbicides, causing necrotic lesions in dicot hosts. Presence of nlp genes has been reported in fungi, oomycetes and bacteria including actinomycetes. In this study, we have over expressed nlp gene from Streptomyces cyaneofuscatus UTMC 2101 in E. coli.
Methods: Whole nlp gene from Streptomyces cyaneofuscatus UTMC 2101 (KF939123) was PCR amplified using a pair of primers which introduced EcoRI and HindIII restriction sites and ligated into identically cleaved pET26b as an expression vector. The final construct was transformed in E. coli BL21 (DE3). PET26b-nlp plasmid was purified and sequenced. A recombinant colony of pET26b-nlp transform ants was inoculated in LB broth with appropriate antibiotic. Final IPTG concentrations of 0.5, 1 and 1.5 mM and incubation temperature of 20, 25, 30 and 37oC were tested to induce the highest recombinant protein production. The periplasmic fraction was collected by osmotic shock method. Both prepared fraction and cell-free culture medium were concentrated with trichloroacetic acid (TCA) precipitation. The periplasmic expression of the recombinant NLP was approved by SDS-PAGE. Non-recombinant pET26b transformant was used as negative control.
Results: Amplification of the genomic DNA of Streptomyces cyaneofuscatus UTMC 2101 with specific nlp primers, resulted in formation of a 828 bp long ORF, encoding for a putative protein with 275 amino acids. The deduced amino acid sequence was predicted to have molecular mass of 29.85 kDa and 90% identity with necrosis inducing factor from Streptomyces flavissimus. Investigating different IPTG concentration and incubation temperature revealed that 1mM IPTG at 20oC is the optimum condition for soluble periplasmic nlp expression. No significant amount of recombinant protein was detected in TCA- precipitated cell-free culture medium. In biological assay of the periplasmic fraction, tobacco leaves necrosis was observed within 48 h after the first spray of non-concentrated periplasmic fraction of pET26b-nlp transform ants, while the leaves remained intact during treatments with periplasmic fraction of the mentioned negative controls.
Conclusion: The obtained data of the current study provided valuable insights into developing a cost-effective bio herbicide to manage dicot weeds especially in monocots farmland using the crude periplasmic fraction of the recombinant E. coli.

 
Keyword(s): PERIPLASMIC EXPRESSION, BIO HERBICIDE, STREPTOMYCES CYANEOFUSCATUS
 
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