Bacterial expression and characterization of chicken apolipoprotein A-I

Protein Expression and Purification
Robert S KissRobert O Ryan

Abstract

Apolipoprotein (apo) A-I is a 28-kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism. It is widely distributed among animal species and is rich in alpha-helical secondary structure. Unlike human apoA-I, which aggregates in the absence of lipid, chicken apoA-I is monomeric in the lipid-free state. To take advantage of this physical characteristic, a bacterial expression system for production of recombinant chicken apoA-I has been developed. The cDNA-encoding chicken apoA-I was cloned into the pET expression vector under the regulation of the lac operon and transformed into Escherichia coli. Recombinant apoA-I protein recovered from the soluble fraction of the bacterial cell pellet was purified to greater than 95% homogeneity by reversed-phase high-performance liquid chromatography. Although immunoblot analysis confirmed the identity of the overexpressed protein, its migration on denaturing polyacrylamide gel electrophoresis was slower than its natural counterpart. To determine if the vector-encoded 18 residue pelB N-terminal leader sequence was not cleaved by the bacterial leader peptidase, isolated recombinant chicken apoA-I was incubated with exogenous leader peptidase. This treatment resulted in an...Continue Reading

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Citations

Aug 26, 1998·Biochimica Et Biophysica Acta·P M WeersR O Ryan

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