Cloning, Expression and Purification of the Low-Complexity Region of RanBP9 Protein
Document Type
Article
Publication Date
8-1-2020
Department
Chemistry and Biochemistry
School
Mathematics and Natural Sciences
Abstract
Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.
Publication Title
Protein Expression and Purification
Volume
172
Recommended Citation
Dhakal, S.,
Sapkota, K.,
Huang, F.,
Rangachari, V.
(2020). Cloning, Expression and Purification of the Low-Complexity Region of RanBP9 Protein. Protein Expression and Purification, 172.
Available at: https://aquila.usm.edu/fac_pubs/17351