Expression and purification of mutated hpn protein, and studies of function of cysteine residues in hpn

Abstract

Hpn is a small nickel-storage protein in Helicobacter pylori. Consisting of 60 amino acids, it is rich in histidine (28 histidine residues), glutamate, glycine and serine residues. There are also four cysteine residues that may play important roles in its structure and function. Our previous study shows that Hpn exists in equilibrium among different multimeric forms in solution, which may be due to intermolecular disulfide bonds formation via cysteine residues. In the present study, we replaced the four-cysteine residues with alanine to abolish disulfide bond formation. Growth assay showed that Escherichia coli BL21(DE3) transformed with mutant hpn gene can grow similarly as the cells transformed with wild type hpn gene under high nickel concentration, suggesting that the mutant hpn protein can still protect the cells against nickel toxicity. This result implies that the mutated protein has similar nickel-binding capacity as the wild type hpn in vivo. The mutant protein was over-expressed and purified. I show that the mutated protein still aggregates, and aggregation state is in equilibrium. Therefore, the underlying mechanism of hpn protein aggregation may not only result from intermolecular disulfide bond formation, but also from the interactions among other amino acids, such as histidines.