Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization
| Autor: | NICOLE MOGUILEVSKY, CORNELIS ROOBOL, ROSETTE LORIAU, JEAN-PAUL GUILLAUME, PAUL JACOBS, ALFREDO CRAVADOR, ALBERT HERZOG, LÉON BROUWERS, ALAIN SCARSO, PASCAL GILLES, LEIF HOLMQUIST, LARS A. CARLSON, ALEX BOLLEN |
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| Jazyk: | angličtina |
| Rok vydání: | 1989 |
| Předmět: |
Molecular Sequence Data
Restriction Mapping Biology medicine.disease_cause Peptide Mapping Biochemistry law.invention Phosphatidylcholine-Sterol O-Acyltransferase chemistry.chemical_compound law Complementary DNA Escherichia coli Genetics medicine Humans Amino Acid Sequence Cloning Molecular Protein Precursors Molecular Biology Peptide sequence Apolipoproteins A chemistry.chemical_classification Expression vector Methionine Apolipoprotein A-I Base Sequence cDNA library DNA Molecular biology Recombinant Proteins Amino acid Liver chemistry Recombinant DNA Plasmids |
| Zdroj: | Repositório Científico de Acesso Aberto de Portugal Repositório Científico de Acesso Aberto de Portugal (RCAAP) instacron:RCAAP |
| Popis: | A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product. |
| Databáze: | OpenAIRE |
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