title: Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.
abstract: The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.
authors.affiliation: Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Upadhyay
authors.forename: Aruna
authors.initials: A
authors.lastname: Murmu
authors.forename: Anupam
authors.initials: A
authors.lastname: Singh
authors.forename: Amulya K
authors.initials: AK
authors.lastname: Panda
mesh_terms: D000682:Amyloid / Q000378:metabolism; D001215:Asparaginase / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D011485:Protein Binding; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism*; D012995:Solubility; D013997:Time Factors
chemical_list: D000682:Amyloid; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone; D019286:Endopeptidase K; D001215:Asparaginase