Heterologous gene expression in a membrane-protein-specific system.
We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.
Turner
G J
GJ
Department of Physiology & Biophysics, University of Miami School of Medicine, Miami, Florida, 33101, USA. gturner@chroma.med.miami.edu
Reusch
R
R
Winter-Vann
A M
AM
Martinez
L
L
Betlach
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D001436:Bacteriorhodopsins / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D011487:Protein Conformation; D018717:Purple Membrane / Q000737:chemistry; D051381:Rats; D051964:Receptors, Mating Factor; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000235:genetics; D015003:Yeasts / Q000737:chemistry
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D051964:Receptors, Mating Factor; D011976:Receptors, Muscarinic; D018000:Receptors, Peptide; D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C484340:SMYD1 protein, human; D014157:Transcription Factors; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D001221:Aspartate Carbamoyltransferase