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Biotechnology and Applied Biochemistry (2002) 35, (91–105) (Printed in Great Britain)

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Review

Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli

Per Jonasson*†, Sissela Liljeqvist*, Per-Åke Nygren* and Stefan Ståhl*¹

*Department of Biotechnology, Kungliga Tekniska Högskolan, Stockholm, Sweden, and †AstraZeneca Biotech Laboratory, EST-Bio, R&D Södertälje, Sweden

Key words: affibody, affinity tag, fusion protein, in vitro refolding, soluble expression.

Abbreviations used: IPTG, isopropyl b-D-thiogalactopyranoside; SPA, Staphylococcus aureus Protein A; RBS, ribosomal binding sites; ZZ, IgG-binding domains derived from staphylococcal protein A; BB, albumin-binding protein from streptococcal protein G; RSV, respiratory syncytial virus; EBA, expanded-bed adsorption; IMAC, immobilized-metal-ion-affinity chromatography.

¹To whom correspondence should be sent, at the following address: Center for Molecular Biotechnology, Department of Biotechnology, SCFAB, KTH, SE-106 91 Stockholm, Sweden (e-mail stefans@biochem.kth.se).

Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-place procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.

Received 1 November 2001; accepted 18 November 2001

Portland Press Ltd © 2002