Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

Arun K Upadhyay,Amulya K Panda,Aruna Murmu,Anupam Singh,Christophe Herman

doi:10.1371/journal.pone.0033951

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.

Highlights

Inclusion bodies (IBs) are formed during high level expression of heterologous proteins in E. coli [1,2]
Proteinase K degradation profiles of asparaginase and human growth hormone (hGH) inclusion bodies To understand the differential physical properties of different inclusion body aggregates, proteolytic susceptibilities of the recombinant hGH and asparaginase inclusion bodies expressed in E. coli were analyzed
Asparaginase IBs solubilized in 3 M urea and refolded by pulsatile dilution method resulted in bioactive protein

Summary

Introduction

Inclusion bodies (IBs) are formed during high level expression of heterologous proteins in E. coli [1,2]. Aggregation of proteins during high level expression is generally attributed to high local concentration of naive polypeptide chains emerging from ribosomes, inefficient folding and unavailability of chaperones [5,6]. These factors lead to the formation of partially folded or misfolded protein intermediates in cytoplasm. The formation of insoluble aggregates of recombinant proteins during high level expression is driven by the association of partially folded or misfolded intermediates. These aggregates have been thought to be resistant to proteases and solubilized only in high molar concentration of chaotropes. It has been reported that inclusion body aggregates can be solubilized using mild solubilization agent without disturbing the native-like secondary structures of proteins [32,33,34]

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