Compartmentalization of mammalian proteins produced in Escherichia coli.
We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.
Rosenwasser
T A
TA
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Hogquist
K A
KA
Nothwehr
S F
SF
Bradford-Goldberg
S
S
Olins
P O
PO
Chaplin
D D
DD
Gordon
J I
JI
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics*; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000378:metabolism
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D007375:Interleukin-1; D008074:Lipoproteins; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins