title: Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein.
abstract: To facilitate structure-function studies of Manduca sexta apolipophorin III (apoLp-III), its nucleotide coding sequence was cloned from a fat body cDNA library by in vitro DNA amplification. The amplification product was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein production by immunoblotting with anti-apoLp-III serum. Data obtained indicated the presence of apoLp-III in both cell lysates and media of cell cultures harboring the apoLp-III-pET construct but not in cells containing the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. Verification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of alpha-helical secondary structure with a further induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that recombinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinant apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluorescence properties of recombinant apoLp-III revealed the presence of a noncovalently associated fluorescent contaminant that was effectively removed by reverse phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
authors.forename: R O
authors.initials: RO
authors.lastname: Ryan
authors.forename: D
authors.initials: D
authors.lastname: Schieve
authors.forename: M
authors.initials: M
authors.lastname: Wientzek
authors.forename: V
authors.initials: V
authors.lastname: Narayanaswami
authors.forename: K
authors.initials: K
authors.lastname: Oikawa
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: L B
authors.initials: LB
authors.lastname: Agellon
mesh_terms: D000818:Animals; D001053:Apolipoproteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli; D017344:Genes, Insect; D050356:Lipid Metabolism; D018402:Manduca; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D001053:Apolipoproteins; D011994:Recombinant Proteins