Expression, purification, and refolding of mouse islet neogenesis associated protein-related protein for NMR studies.
Islet neogenesis associated protein-related protein (INGAPrP) is thought to be involved in the differentiation of non-insulin-producing cells to insulin-secreting cells. INGAPrP is a mouse gene product that has a 72% identical amino acid sequence to a known islet-generating factor, hamster islet-neogenesis-associated protein (INGAP), which acts by differentiating pancreatic ductal cells into beta-cells. The three-dimensional structure of these proteins is unknown. The structure would provide information about the conformation of the active portion of INGAP, the so-called INGAP pentadecapeptide, leading to a well-defined target for rational drug design. An efficient procedure for the production of INGAPrP would facilitate the process of structure determination. We have successfully produced and isolated (15)N-labeled INGAPrP by expression in Escherichia coli Rosetta (DE3) cells in Spectra-9 media followed by a two-step purification and refolding protocol. The hexahistidine tag engineered at the N-terminus of the protein is used in the first step for standard immobilized-metal affinity chromatography purification under denaturing conditions. The secondary purification step utilizes a gel permeation chromatography column, producing homogeneous INGAPrP as well as refolding the protein. To verify that the protein was folded, we performed a (1)H-(15)N HSQC NMR experiment that showed excellent dispersion of signals, indicative of a folded protein. We also performed circular dichroism experiments, which demonstrated the presence of secondary structure. In summary, we report the first expression and isolation of INGAPrP, as well as demonstrate that our method produces a folded protein, which is necessary for structure determination.
Kulis
Michael D
MD
School of Chemistry and Biochemistry, Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, 30332, USA.
Shuker
Suzanne B
SB
D000595:Amino Acid Sequence; D000818:Animals; D015870:Gene Expression; D007515:Islets of Langerhans / Q000378:metabolism; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D000073718:Pancreatitis-Associated Proteins; D017510:Protein Folding; D020673:Protein Renaturation; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
D000073718:Pancreatitis-Associated Proteins; D011506:Proteins; C000612141:REG3A protein, human; C490252:Reg3d protein, mouse