High-level secretion of human apolipoprotein E produced in Escherichia coli: use of a secretion plasmid containing tandemly polymerized ompF-hybrid gene.
A gene encoding the mature form of human apolipoprotein E (h-apoE) was fused to the secretion signal coding sequence of the Escherichia coli major outer membrane protein F (ompF) which was preceded by a consensus Shine-Dalgarno sequence. Two copies of this hybrid gene were inserted tandemly into an expression vector and expressed in E. coli under the transcriptional control of two tac promoters regulated by lac repressors. By the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to the growth media, cells synthesized h-apoE at the level of 27.2 micrograms per A600 and up to 22% of the total cellular protein. The h-apoE produced by E. coli was processed precisely, secreted into the periplasmic space and formed protein aggregates there. However, despite aggregation, they were easily dissolved in water and actively formed protein-lipid complexes with dimyristoyl phosphatidyl choline (DMPC). These results demonstrated that E. coli cells are able to synthesize and secrete a large amount of active h-apoE using a prokaryotic signal sequence.
Shibui
T
T
Biosciences Laboratory, Mitsubishi Kasei Corporation, Kanagawa, Japan.
Uchida-Kamizono
M
M
Okazaki
H
H
Kondo
J
J
Murayama
S
S
Morimoto
Y
Y
Nagahari
K
K
Teranishi
Y
Y
D000595:Amino Acid Sequence; D001057:Apolipoproteins E / Q000096:biosynthesis / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism
D001057:Apolipoproteins E; D001425:Bacterial Outer Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside