Isolation of point mutations that affect the folding of the H- chain of human ferritin in E. coli

A. Luzzago, G. Cesareni

Research output: Contribution to journalArticlepeer-review

Abstract

We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process. Apo-ferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures. We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding-assembly process is altered or completely impaired. The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the α-peptide of β-galactosidase. In the wild type situation, the α-peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function. We show that by selecting for mutations that restore β-galactosidase activity we are able to identify ferritin mutations that affect the folding - assembly process. The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy-terminal E helix in the 4-fold axis.

Original languageEnglish
Pages (from-to)569-576
Number of pages8
JournalEMBO Journal
Volume8
Issue number2
Publication statusPublished - 1989

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

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