Over expression and purification of T7 tail fibre gp17 in E. Coli BL21 for raising antibodies in rabbit

T7 phage is used extensively for the construction of peptide display library. However, identifying specific phage displaying peptide of interest requires antibody to capture T7-phage through tail-fibre without interfering with functionality of the displayed peptide. To raise high quality antibodies,...

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Bibliographic Details
Published in:Indian journal of clinical biochemistry 2022-05, Vol.33 (S1), p.S37
Main Authors: Kumarasamy, J, Dhekale, Yogesh, Kulkarni, Savita
Format: Article
Language:English
Subjects:
Animal genetic engineering
Antibodies
Escherichia coli
Viral antibodies
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Summary:T7 phage is used extensively for the construction of peptide display library. However, identifying specific phage displaying peptide of interest requires antibody to capture T7-phage through tail-fibre without interfering with functionality of the displayed peptide. To raise high quality antibodies, it is imperative to have pure T7 tail-fibre protein which was overexpressed in E. Coli, purified and used for immunization. C-terminal domain of the bacteriophage T7 tail-fibre protein gp17 was cloned in pET-30a + by van Raaij MJ., et al., and was gifted to us by Prof. Jose L. Carrascosa., Centro Nacional de Biotecnologia, Spain. E. Coli BL21(DE3) was transformed with fibre construct in pET-30a + vector by electroporation. The transformed colonies were grown and selected on kanamycin plates. Over expression was induced with 1 mM IPTG overnight at 16[degrees]C. Harvested cells were lysed using bugbuster master-mix, over expression was confirmed on SDS-PAGE. Further, T7-fibre protein was purified on NiNTA-agarose eluted with imidazole on step-gradient. 1 mM IPTG was optimum to overexpress the T7 tail fibre protein. Maximum elution was at 200 mM of imidazole. However, pure fraction (single band) was obtained with 300 and 400 mM confirmed by silver staining. Purified protein was used for rabbit immunization. T7 fibre protein was overexpressed, purified and pure protein was used for raising polyclonal antiserum in rabbit. KEY WORDS: T7 tail fiber, gp17, NiNTA purification
ISSN:0970-1915