Academic journal article Iranian Journal of Public Health

Recombinant Nlp Production from Streptomyces Cyaneofuscatus Utmc 2101in E. Coli

Academic journal article Iranian Journal of Public Health

Recombinant Nlp Production from Streptomyces Cyaneofuscatus Utmc 2101in E. Coli

Article excerpt

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 Eco RI 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 se- quenced. A recombinant colony of pET26b-nlp trans- formants was inoculated in LB broth with appropriate antibi- otic. Final IPTG concentrations of 0.5, 1 and 1.5 mM and incubation temperature of 20, 25, 30 and 37 °C 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 trans- formant 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. …

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