Báo cáo khoa học: Concepts and tools to exploit the potential of bacterial inclusion bodies in

MINIREVIEW

Concepts and tools to exploit the potential of bacterial

inclusion bodies in protein science and biotechnology

Pietro Gatti-Lafranconi1,*, Antonino Natalello2,*, Diletta Ami2

, Silvia Maria Doglia2 and Marina Lotti2

1 Department of Biochemistry, University of Cambridge, UK

2 Department of Biotechnology and Biosciences, State University of Milano-Bicocca, Italy

Protein aggregation in the bacterial

cytoplasm: regulation, override and

effects

It is estimated that the global macromolecule concen￾tration in the Escherichia coli cytoplasm is around

200–400 gÆL)1 and that macromolecules occupy 20–

30% of the total cytoplasmic volume [1,2]. Individual

proteins are represented at relatively low concentration

(nm to lm) but in the cytoplasm this translates into

the distance between any two molecules having the

same dimensions as proteins themselves [3]. Crowding

increases non-specific, attractive and electrostatic inter￾actions and modifies diffusion rates, with detrimental

effects on the behaviour of all macromolecules [4]. In

these conditions, folding becomes a kinetic race against

aggregation: although the native state is thermodynam￾ically favoured [5], aggregation can trap folding inter￾mediates into non-native folding landscapes that,

in the absence of further control mechanisms, would

Keywords

aggregation; amyloid-like structures;

biocatalysis; electron and optical

microscopies; fourier transform infrared

spectroscopy; inclusion bodies; IB structural

properties; native-like conformation;

recombinant proteins; stress response

Correspondence

S. M. Doglia, M. Lotti, Department of

Biotechnology and Biosciences, State

University of Milano-Bicocca, Piazza della

Scienza 2, 20126 Milano, Italy

Fax: +39 02 64483565

Tel: +39 02 64483459

E-mail: [email protected];

[email protected]

*These authors contributed equally to this

work

(Received 28 January 2011, revised 20

March 2011, accepted 5 April 2011)

doi:10.1111/j.1742-4658.2011.08163.x

Cells have evolved complex and overlapping mechanisms to protect their

proteins from aggregation. However, several reasons can cause the failure

of such defences, among them mutations, stress conditions and high rates

of protein synthesis, all common consequences of heterologous protein pro￾duction. As a result, in the bacterial cytoplasm several recombinant pro￾teins aggregate as insoluble inclusion bodies. The recent discovery that

aggregated proteins can retain native-like conformation and biological

activity has opened the way for a dramatic change in the means by which

intracellular aggregation is approached and exploited. This paper summa￾rizes recent studies towards the direct use of inclusion bodies in biotechnol￾ogy and for the detection of bottlenecks in the folding pathways of specific

proteins. We also review the major biophysical methods available for

revealing fine structural details of aggregated proteins and which informa￾tion can be obtained through these techniques.

Abbreviations

DAAO, D-amino acid oxidase; GFP, green fluorescent protein; IB, inclusion body; TF, trigger factor.

2408 FEBS Journal 278 (2011) 2408–2418 ª 2011 The Authors Journal compilation ª 2011 FEBS