HETEROLOGOUS EXPRESSION IN ESCHERICHIA-COLI OF NATIVE AND MUTANT FORMS OF THE MAJOR INTRINSIC PROTEIN OF RAT EYE LENS (MIP26)

The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence dis played 89% nucleotide identity and 95% identity at the amino acid leve l with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Ce ll 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for exp ression in Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic o ctapeptide tail (FLAG) was fused to either the Nor C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identfied by using a monoclonal ant ibody corresponding to the FLAG peptide located at the termini of MIP2 6. Immunefluorescently labelled monoclonal antibody was used to determ ine the localization of MIP26 in the cytoplasmic membrane. The majorit y of the protein was integrated into cell plasma membrane. MIP26 was e xtracted with n-octyl beta-D-glucopyranoside and then purified on an a ffinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at t he C-terminus, which has been shown in other proteins to be particular ly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn(244) --> Asp) near the C-term inus. The mutation was cofirmed by DNA sequencing. The mutant MIP26 pr otein was also expressed in E. coli and incorporated predominantly int o the cytoplasmic membrane.