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 displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly 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 (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane.
Dilsiz
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Wolfson Laboratory, School of Animal and Microbial Sciences, University of Reading, Whiteknights, U.K.
Crabbe
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D020346:Aquaporins; D018076:DNA, Complementary; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C030328:aquaporin 0