Hydrophobicity Engineering to Increase Solubility and Stability of a Recombinant Protein from Respiratory Syncytial Virus
| Autor: | Laurent Mignard, Mathias Uhlén, Stefan Ståhl, Maria Murby, Thien Ngoc Nguyen, Ultan F. Power, Hans Binz, Elisabet Samuelsson |
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| Rok vydání: | 1995 |
| Předmět: |
Circular dichroism
HN Protein Circular Dichroism Recombinant Fusion Proteins Molecular Sequence Data Mutagenesis (molecular biology technique) Enzyme-Linked Immunosorbent Assay Protein engineering Biology Protein Engineering Biochemistry Fusion protein Inclusion bodies Respiratory Syncytial Viruses Gene product Viral Proteins Drug Stability Solubility Viral Envelope Proteins Amino Acid Sequence Cysteine |
| Zdroj: | European Journal of Biochemistry. 230:38-44 |
| ISSN: | 1432-1033 0014-2956 |
| Popis: | Site-directed mutagenesis has been employed to engineer the hydrophobic properties of a 101-amino-acid fragment from the human respiratory syncytial virus (RSV) major glycoprotein (G protein). When this protein was produced in Escherichia coli, more than 70% of the gene product was found as inclusion bodies, and the product recovered from the soluble fraction was severely degraded. Substitution of two cysteine residues for serine residues, did not significantly change the solubility or stability of the gene product. In contrast, a dramatic increase in both solubility and stability was achieved by multiple engineering of hydrophobic phenylalanine residues. As compared to the non-engineered protein, the fraction of soluble protein in vivo could be increased from 27% to 75%. Surprisingly, this effect was accompanied by a remarkable increase in stability. The in vitro solubility of the purified gene products was similarly increased approximately fivefold. Structural studies using circular dichroism suggest that the two engineered fragments have a distribution of secondary-structure elements similar to the non-engineered fragment. In addition, the two engineered G-protein variants were demonstrated to be at least in part antigenically authentic to the non-engineered gene product. These results demonstrate that engineering of hydrophobic residues can be used as a tool to increase the solubility and proteolytic stability of poorly soluble and labile proteins. |
| Databáze: | OpenAIRE |
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