Experimental and theoretical study of IBC domain from human IP3R2; molecular cloning, bacterial expression and protein purification

Autor: Farangis Ataei, Marzieh Nakhaei Amroudie
Rok vydání: 2019
Předmět:
Models
Molecular
Protein Conformation
alpha-Helical
DNA
Complementary
Genetic Vectors
Gene Expression
lac operon
Inositol 1
4
5-Trisphosphate
02 engineering and technology
Molecular cloning
Ligands
Biochemistry
Mice
03 medical and health sciences
chemistry.chemical_compound
Structural Biology
Protein purification
Escherichia coli
Animals
Humans
Inositol 1
4
5-Trisphosphate Receptors
Protein Interaction Domains and Motifs
Amino Acid Sequence
Cloning
Molecular
Molecular Biology
030304 developmental biology
Cloning
0303 health sciences
Binding Sites
Expression vector
Sequence Homology
Amino Acid
Computational Biology
General Medicine
021001 nanoscience & nanotechnology
Ligand (biochemistry)
Recombinant Proteins
Protein Structure
Tertiary
chemistry
Protein Conformation
beta-Strand
RNA extraction
0210 nano-technology
Sequence Alignment
DNA
Protein Binding
Zdroj: International Journal of Biological Macromolecules. 124:1321-1327
ISSN: 0141-8130
DOI: 10.1016/j.ijbiomac.2018.09.117
Popis: IP3 is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+-release from intracellular stores. IP3 binds to intracellular IP3-receptor (IP3R) and induces conformational change within the ligand-binding domain which regulates Ca2+-release; hence, both IP3 and IP3R are key components of the signal transduction mechanism. Here we present cDNA cloning of IP3-binding core (IBC) domain encoding only residues 224–604 of human IP3R type 2 that binds to IP3 with high affinity. RNA extraction, RT-PCR, PCR and cloning were carried out, and then the cloned DNA was checked by sequencing. Thereafter, expression vector pET-28a harboring the correct gene was transformed into different E. coli (DE3) strains and investigated its protein expression under various conditions. Finally, the IBC expression was induced at 20 °C for 20 h into BL21 strain at LB medium with 4 mM lactose and 0.5 mM IPTG, and then confirmed by western blotting. After protein purification, structural study was recorded in absence and presence of its ligand. Far-CD and intrinsic fluorescence spectra analysis of the purified protein with and without IP3 ligand showed change in secondary and tertiary IBC structure. Moreover, bioinformatics study demonstrated that the ligand binding site residues R269, K508 and R511 are conserved.
Databáze: OpenAIRE
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