HYDROPHOBICITY ENGINEERING TO INCREASE SOLUBILITY AND STABILITY OF A RECOMBINANT PROTEIN FROM RESPIRATORY SYNCYTIAL VIRUS

Site-directed mutagenesis has been employed to engineer the hydrophobi c properties of a 101-amino-acid fragment from the human respiratory s yncytial virus (RSV) major glycoprotein (G protein). When this protein was produced in Escherichia coli, more than 70% of the gene product w as found as inclusion bodies, and the product recovered from the solub le fraction was severely degraded. Substitution of two cysteine residu es for serine residues, did not significantly change the solubility or stability of the gene product. In contrast, a dramatic increase in bo th solubility and stability was achieved by multiple engineering of hy drophobic phenylalanine residues. As compared to the non-engineered pr otein, 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 p roducts was similarly increased approximately fivefold. Structural stu dies using circular dichroism suggest that the two engineered fragment s have a distribution of secondary-structure elements similar to the n on-engineered fragment. In addition, the two engineered G-protein vari ants were demonstrated to be at least in part antigenically authentic to the non-engineered gene product. These results demonstrate that eng ineering of hydrophobic residues can be used as a tool to increase the solubility and proteolytic stability of poorly soluble and labile pro teins.