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Recombinant Human Proapolipoprotein A-I: Experimental Strategies for the Production of an Authentic Molecule

  • Conference paper
Human Apolipoprotein Mutants III

Part of the book series: NATO ASI Series ((ASIH,volume 73))

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Summary

A clone coding for human proapolipoprotein A-I (proapo A-I) was isolated from a human liver cDNA library. Expression of recombinant proapo A-I in the cytoplasm of E.coli or baculovirus-infected insect cells led to the production of a polypeptide which was characterized in terms of apparent molecular mass, isoelectric point, chemical and enzymatic peptide mapping. In addition, the recombinant protein was assayed in vitro for the stimulation of the enzyme lecithin:cholesterol acyltransferase. The data showed that rec proapo A-I was produced at high level in E.coli and in insect cells as a stable protein having properties identical to those of the natural product. However, none of these expression systems was able to remove the N-terminal methionine from the recombinant protein. Several methods were thus compared for the production of authentic, N-terminal methionine-free proapo A-I in engineered Escherichia coli bacteria. Secretion, mediated by the OmpA signal peptide, appeared the best approach, yielding authentic proapo A-I from engineered bacteria. At last, in view of the potential therapeutical application of rec proapo A-I, assessing of DNA contamination in purified batches of the protein was performed using a quantitative polymerase chain reaction assay.

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Author information

Authors and Affiliations

  1. Applied Genetics, U.L.B, rue de l’Industrie 24, B-1400, Nivelles, Belgium

    N. Moguilevsky, F. Varsalona, J.-P. Guillaume & A. Bollen

  2. UCB-Pharma, chemin du Foriest, B-1420, Braine-l’Alleud, Belgium

    C. Roobol

Authors
  1. N. Moguilevsky
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  2. F. Varsalona
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  3. J.-P. Guillaume
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  4. C. Roobol
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  5. A. Bollen
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Editor information

Editors and Affiliations

  1. Center E. Grossi Paoletti, Institute of Pharmacological Sciences, University of Milano, Via Balzaretti 9, 20133, Milano, Italy

    Cesare R. Sirtori  & Guido Franceschini  & 

  2. Molecular Disease Branch, National Institutes of Health, NHLBI, 20205, Bethesda, MD, USA

    Bryan H. Brewer Jr.

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© 1993 Springer-Verlag Berlin Heidelberg

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Moguilevsky, N., Varsalona, F., Guillaume, JP., Roobol, C., Bollen, A. (1993). Recombinant Human Proapolipoprotein A-I: Experimental Strategies for the Production of an Authentic Molecule. In: Sirtori, C.R., Franceschini, G., Brewer, B.H. (eds) Human Apolipoprotein Mutants III. NATO ASI Series, vol 73. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-84634-2_14

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  • DOI: https://doi.org/10.1007/978-3-642-84634-2_14

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-84636-6

  • Online ISBN: 978-3-642-84634-2

  • eBook Packages: Springer Book Archive

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