Truncation of C-Terminal Intrinsically disordered region of mycobacterial Rv1915 facilitates production of “Difficult-to-Purify” recombinant drug target

Monika Antil (1) , Sebastien G. Gouin (2) , Vibha Gupta (1)

1 JIIT - Jaypee Institute of Information Technology
2 CORAIL - Catalysis, ORganometallic chemistry And synthesIs of Ligands

Availability of purified drug target is a prerequisite for its structural and functional characterization. However, aggregation of recombinant protein as inclusion bodies (IBs) is a common problem during the large scale production of overexpressed protein in heterologous host. Such proteins can be recovered from IB pool using some mild solubilizing agents such as low concentration of denaturants or detergents, alcohols and osmolytes. This study reports optimization of solubilization buffer for recovery of soluble and biologically active recombinant mycobacterial Rv1915/ICL2a from IBs. Even though the target protein could be solubilized successfully with mild agents (sarcosine and βME) without using denaturants, it failed to bind on Ni-NTA resin. The usual factors such as loss of His6-tag due to proteolysis, masking of the tag due to its location or protein aggregation were investigated, but the actual explanation, provided through bioinformatics analysis, turned out to be presence of intrinsically disordered protein regions (IDPRs) at the C-terminus. These regions due to their inability to fold into ordered structure may lead to non-specific protein aggregation and hence reduced binding to Ni-NTA affinity matrix. With this rationale, 90 residues from the C-terminal of Rv1915/ICL2 were truncated, the variant successfully purified and characterized for ICL and MICL activities, supporting the disordered nature of Rv1915/ICL2a C-terminal. When a region that has definite structure associated in some mycobaterial strains such as CDC 1551 and disorder in others for instance Mycobacterium tuberculosis H37Rv, it stands to reason that larger interface in the later may have implication in binding to other cellular partner.

Mots clés: Mycobacterium tuberculosis H37Rv; Inclusion bodies; Isocitrate lyase 2; Solubilization and IDPRs

Domaines: Biotechnologies

Monika Antil, Sebastien G. Gouin, Vibha Gupta. Truncation of C-Terminal Intrinsically disordered region of mycobacterial Rv1915 facilitates production of “Difficult-to-Purify” recombinant drug target. Frontiers in Bioengineering and Biotechnology, 2020, 8, pp.522. ⟨10.3389/fbioe.2020.00522⟩. ⟨hal-03375267⟩