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Published on: May 18, 2021
Molecular cloning, expression and generation of a polyclonal antibody specific for RNPS1
Bhagyashree Deka1, Kusum Kumari Singh2
1Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, 781039, Guwahati, Assam, India.
Background:
RNA-binding protein with serine-rich domain 1 (RNPS1) is a member of a splicing-dependent mega Dalton protein complex or exon junction complex (EJC). During splicing, RNPS1 acts as a protector of global transcriptome integrity by suppressing the usage of cryptic splice sites. Additionally, RNPS1 functions in almost all stages of mRNA metabolism, including constitutive splicing, alternative splicing, translation and nonsense-mediated mRNA decay (NMD). The aim of the present study was to generate a highly specific polyclonal antibody against human RNPS1.
Methods And Results:
A plasmid, pHis-TEV-RNPS1, has been constructed to overexpress recombinant RNPS1 (22-305 amino acids) by cloning the nucleotide sequence downstream of an N-terminal His-tag in the parent plasmid pHis-TEV. The recombinant plasmid was then transformed into Rosetta and expression was induced using IPTG. The His-tagged RNPS1 protein was purified using Ni-NTA affinity chromatography. The rabbit antiserum was then obtained by immunizing rabbits with the purified recombinant RNPS1 protein. The antiserum was further purified by antigen-immunoaffinity chromatography. The sensitivity and the specificity of the polyclonal antibody were assessed by enzyme-linked immunosorbent assay (ELISA) and knockdown assay. ELISA demonstrated that the antibody has a high binding affinity for RNPS1 and the usable titre is 1:2000.
Conclusion:
The antibody detected RNPS1 in human, mouse cell lines and rat tissue in Western blot. Importantly, the antibody efficiently detected the decrease in RNPS1 expression in siRNA induced knockdown assay, indicating the specificity of the antibody. The polyclonal antibody against RNPS1 will be a useful tool for performing further functional studies on RNPS1.
