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Table 1 Protein engineering strategies to reduce aggregation or derived effects during either production or administration, illustrated by representative examples.

From: Post-production protein stability: trouble beyond the cell factory

Protein engineering strategy

Result

Protein

Reference

Improving protein folding during production

   

Cys→Ser point mutations

Reduced aggregation, enhanced proteolytic stability

bFGFa

[100]

Point mutations in an hydrophobic stretch

Reduced aggregation

11 beta-HSD1

[101]

Directed evolution/point mutations

Reduced aggregation

Cytochrome P450sca-2

[102]

Fusion of SUMO tag

Improved refolding

Fgf15

[103]

Polycationic amino acid tag fusion

Reduced aggregation

Candida antarctica lipase B

[104]

Fusion to polylysines or polyarginines

Reduced aggregation

BPTI-22

[105]

Fusion to MBP

Reduced aggregation

Ribonuclease inhibitor

[106]

Fusion to GrpE

Reduced aggregation

hIL-3

[107]

Fusion to NusA

Reduced aggregation, enhanced proteolytic stability

E8R viral protein

[108]

Improving protein folding, stability and performance during administration

   

Single amino acid substitution

Inhibited oligomer formation; enhanced bioavailability

Insulin Aspart ®

[109]

Single amino acid substitution

Improved folding

INF-β-1b (Betaferon ®)

[110]

N-terminal peptide deletion

Enhanced stability

KGF

[111]

Fusion with albumin

Extended half-life

Albinterferon α-2b

[76]

Fusion with transferrin

Enhanced gastrointestinal adsorption

hGH

[75]

Artificial consensus protein sequence

Enhanced activity

Interferon αcon-1 (Infergen ®)

[112]

Fusion of a HIV Tat segment

Enhanced solubility

p53

[113]

Fusion of a HIV Tat protein and ODD

Enhanced stability and activity in hypoxic tumor cells

Casp-3

[114]

Fusion of a HIV Tat protein and sequence modification

Cell penetration and selective activation in HIV-infected cells

Casp-3

[115]

Ligand incorporation (mainly antibody fragments)

Enhanced stability and bioavailbility

IL-2

[71]

  1. a Abbreviations are: 11 beta-HSD1, 11 beta-Hydroxysteroid dehydrogenase type 1; aFGF, acidic fibroblast growth factor; bFGF: Fgf15, Fibroblast growth factor 15; BPTI-22, Bovine pancreatic trypsin inhibitor variant 22; Casp-3, caspase 3; HIV, human immunodeficiency virus; hFGF, Human basic fibroblast growth factor; BSA, bovine serum albumin; HAS, human serum albumin; hGH, human growth hormone; hIL-3, human interleukin-3; KGF, keratinocyte growth factor; IL, interleukin; MAGOH, Protein mago nashi homolog; MBP, maltose-binding protein; OOD, oxygen-dependent degradation domain of hypoxia-inducible factor-1alpha; rhDNase, recombinant human DNAse; SUMO, small ubiquitin-related modifier.