PMID: 9192073Jan 1, 1997Paper

Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E

Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
C A FisherR O Ryan

Abstract

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroi...Continue Reading

Citations

Jan 20, 2006·The FEBS Journal·Katherine A RedmondRobert O Ryan
Apr 23, 2009·Protein Science : a Publication of the Protein Society·Arun SivashanmugamQianqian Li
Feb 29, 2008·Protein Science : a Publication of the Protein Society·Wentao ZhaoJianjun Wang
Oct 10, 2007·Analytical Biochemistry·Taichi YamamotoRobert O Ryan
Jul 4, 2001·European Journal of Biochemistry·P M WeersR O Ryan
Sep 24, 1999·Protein Science : a Publication of the Protein Society·D ClaytonR Smith
Apr 24, 2003·The Journal of Biological Chemistry·Vincent RaussensRobert O Ryan

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