Enhancement of the yield of long helical structure of recombinant nucleocapsid protein of Newcastle disease virus

• Published in: Process biochemistry (1991) Vol. 48; no. 2; pp. 267 - 271
• Main Authors: Yap, Chee Fai, Tan, Wen Siang, Sieo, Chin Chin, Tey, Beng Ti
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.02.2013
• Subjects: Avian orthoavulavirus 1; computer software; EDTA (chelating agent); Endogenous protease; Escherichia coli; image analysis; mutants; Newcastle disease virus; Nucleocapsid protein; nucleocapsid proteins; PeptideCutter; Protease inhibitor; proteinase inhibitors; proteolysis; serine proteinases; synergism; Western blotting; Newcastle disease virus; Endogenous protease; Nucleocapsid protein; Escherichia coli; PeptideCutter; Protease inhibitor
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2013.01.004

► Western blot image analysis was used to predict proteases cleavage sites ofNPΔc375. ► PeptideCutter program was used to identify the protease that attacking NPΔc375. ► Metallo- and serine-proteases were predicted as the protease that attacking NPΔc375. ► EDTA and PMSF were used to inhibit activities of metallo- and serine-proteases. ► The particle assembly property and antigenicity of treated NPΔc375 were retained. A deletion mutant of the nucleocapsid protein (NPΔc375) of Newcastle disease virus self-assembles into a long helical structure when expressed in Escherichia coli. However, the NPΔc375 subjects to proteolytic activity of host cell endogenous proteases during the protein recovery process. Image analysis of Western blots using the Quantity One software was performed to identify the size of the degraded bands and hence the potential proteases cleavage sites were predicted. The data obtained from this image analysis were compared to those identified with the PeptideCutter program; the potential proteases that degrade the NPΔc375 were identified to be mainly the metallo and serine proteases. Combination of ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride at their optimal concentration gave a synergistic effect and increased the NPΔc375 yield by 2.1-fold. The antigenicity and self-assembled long helical structure long helical structure of NPΔc375 were preserved after treatment with the protease inhibitors.