Large Scale Production of Potato Cirus Y Necrotic Strain (PVYN) Coat protein through Expression the CP Gene in E. coli
Large Scale Production of Potato Cirus Y Necrotic Strain (PVYN) Coat protein through Expression the CP Gene in E. coli
A.S. Gamal El din1; Sohair I. El-Afifi2; A. S. Sadik 2; Nashwa M. A. Abd El-Mohsen1; and H. M. Abdelmaksoud1
ABSTRACT
A fragment with a size of 801 bp from the CP gene was amplified from Potato virus YN isolated from potato plants cultivated at Monofyia Governorate. RNA was extracted from virus-infected tissues of D. metel leaves by the use of degenerate primers through polymerase chain reaction (PCR). Large-scale amount of PVY (N-Egypt) coat protein produced by gene expression technique in E. coli through: (I) Insertion of CP gene isolated by RT-PCR into Pinpoint Xa-l Vector by ligation and propagation after transformation process in E. coli. (2) Isolation of plasmid DNA. then used restriction enzymes Bam HI and Bgl II to identify clones containing inserts. To confirm the fragment inserted of CP gene sequence. PCR was performed using specific primers for PVYN CP gene. (3) The gene expression was performed using 100 µM IPTG & 2 µM biotin and incubation for 4h / 37 oC to produce biotinylated protein for E. coli with Mr 22.5 KDa and fusion protein with Mr ≈ 48 KDa consists or biotin tag and CP product or PVYN. (4) The protein was purified by softlink™ soft release avidin resin. About 2.85 mg/ml of the expressed protein was purified from I L of bacterial culture.
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