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Journal of Lipid Research, Vol 36, 1066-1072, Copyright © 1995 by Lipid Research, Inc.
RO Ryan, D Schieve, M Wientzek, V Narayanaswami, K Oikawa, CM Kay and LB Agellon
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)
ARTICLES
Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
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H. A. Garda, E. L. Arrese, and J. L. Soulages Structure of Apolipophorin-III in Discoidal Lipoproteins. INTERHELICAL DISTANCES IN THE LIPID-BOUND STATE AND CONFORMATIONAL CHANGE UPON BINDING TO LIPID* J. Biol. Chem., May 24, 2002; 277(22): 19773 - 19782. [Abstract] [Full Text] [PDF]
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J. Wang, S. M. Gagne, B. D. Sykes, and R. O. Ryan Insight into Lipid Surface Recognition and Reversible Conformational Adaptations of an Exchangeable Apolipoprotein by Multidimensional Heteronuclear NMR Techniques J. Biol. Chem., July 18, 1997; 272(29): 17912 - 17920. [Abstract] [Full Text] [PDF]
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V. Narayanaswami, J. Wang, C. M. Kay, D. G. Scraba, and R. O. Ryan Disulfide Bond Engineering to Monitor Conformational Opening of Apolipophorin III During Lipid Binding J. Biol. Chem., October 25, 1996; 271(43): 26855 - 26862. [Abstract] [Full Text] [PDF]
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