Heterologous expression of bovine and porcine β-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine β-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promot...

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Bibliographic Details
Published in:Journal of biotechnology 2004-04, Vol.109 (1), p.169-178
Main Authors: Invernizzi, Gaetano, Ragona, Laura, Brocca, Stefania, Pedrazzoli, Eros, Molinari, Henriette, Morandini, Piero, Catalano, Maddalena, Lotti, Marina
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Biotechnology
cattle
Cattle - genetics
Cloning, Molecular
Expression
Fundamental and applied biological sciences. Psychology
Gene Expression
genetically engineered microorganisms
lactoglobulins
Lactoglobulins - biosynthesis
Lactoglobulins - genetics
Lactoglobulins - isolation & purification
Magnetic Resonance Spectroscopy
NMR
Pichia - genetics
Pichia - physiology
Pichia pastoris
Promoter Regions, Genetic
protein secretion
protein synthesis
recombinant proteins
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
swine
Swine - genetics
β-Lactoglobulin
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Summary:Bovine and porcine β-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 μg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2003.10.034