BACTERIAL EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF A FUNCTIONAL RECOMBINANT APOLIPOPROTEIN

To facilitate structure-function studies of Manduca sexta apolipophori n III (apoLp-III), its nucleotide coding sequence was cloned from a fa t body cDNA library by in vitro DNA amplification. The amplification p roduct was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein pr oduction by immunoblotting with anti-apoLp-III serum. Data obtained in dicated the presence of apoLp-III in both cell lysates and media of ce ll cultures harboring the apoLp-III-pET construct but not in cells con taining the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. V erification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular di chroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of a-helical secondary structure with a furt her induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that reco mbinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinan t apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluoresc ence properties of recombinant apoLp-III revealed the presence of a no ncovalently associated fluorescent contaminant that was effectively re moved by reverse phase HPLC. Subsequent fluorescence characterization revealed the expected intrinsic quenching of tyrosine fluorescence in buffer. Furthermore, in a manner very similar to the native protein, t yrosine fluorescence in recombinant apoLp-III was greatly enhanced upo n interaction with detergent micelles, indicating an ability to underg o characteristic conformational changes upon lipid interaction. An F14 8L mutant was isolated whose tyrosine fluorescence quantum yield was c onsiderably higher than that observed for natural apoLp-III. In urea d enaturation studies, the F148L apoLp-III was found to be less stable t han wild type apoLp-III. Bacterial expression of full length, soluble and functional apoLp-III offers a useful system to probe the structure -function relationship of this unique apolipoprotein.