Overexpression of higher eukaryotic membrane proteins in bacteria. Novel insights obtained with the liver mitochondrial proton/phosphate symporter.
In order to better understand why higher eukaryotic membrane proteins, in contrast to soluble proteins, are not readily expressed in Escherichia coli, the gene encoding the liver mitochondrial phosphate transporter (H+/Pi symporter) (Ferreira, G. C., Pratt, R. D., and Pedersen, P. L. (1989) J. Biol. Chem. 264, 15628-15633), was subcloned into a plasmid (pFOG402) containing the alkaline phosphatase promoter and leader sequence. Although this system is highly efficient in overexpressing soluble mitochondrial proteins in E. coli, e.g. alpha and beta subunits of the liver ATP synthase, it fails to express the H+/Pi transporter. Expression is not obtained by truncation of the transporter gene from either the 3' or 5' end, by fusing the mature transporter gene to genes encoding either the alpha or beta ATP synthase subunits, or by using different expression plasmids. Significantly, the H+/Pi transporter is overexpressed in E. coli provided its cDNA is first truncated at the 3' end (carboxyl-terminal end) and fused to a cDNA fragment derived from the ATP synthase alpha subunit gene. In fact, progressive deletions from the 3' end of the transporter cDNA produce a ladder of increasingly overexpressed fusion proteins which account from the largest to the smallest for approximately 2.5-14% of the total bacterial cell protein. The minimal truncation necessary from the 3' end is 192 base pairs corresponding to 64 COOH-terminal amino acids. This corresponds to 20% of the transporter and involves removal of one of the six predicted membrane-spanning segments. In a variety of additional experiments designed to define the molecular basis for E. coli's inability to express the complete liver H+/Pi transporter, problems related to cell toxicity and transcription were ruled out. However, in vitro transcription-translation assays revealed that the complete transporter is readily expressed when eukaryotic, but not prokaryotic, ribosomes are present. Significantly, the fused transporter gene (i.e. Pi transporter cDNA truncated at the 3' end + ATP synthase alpha subunit cDNA) is expressed when prokaryotic ribosomes are present. These results support the view that the difficulty in expressing higher eukaryotic membrane proteins in bacteria may be related in some cases to a problem at the level of translation.
Ferreira
G C
GC
Department of Biological Chemistry, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205.
Pedersen
P L
PL
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism*; D028044:Phosphate-Binding Proteins; D014176:Protein Biosynthesis; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D002352:Carrier Proteins; D028044:Phosphate-Binding Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases