Downstream coding region determinants of bacterio-opsin, muscarinic acetylcholine receptor and adrenergic receptor expression in Halobacterium salinarum.
The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum.
Bartus
Cynthia L
CL
Department of Physiology and Biophysics, and the Neuroscience Program, University of Miami School of Medicine, PO Box 016430, 1600 NW 10th Avenue, Miami, FL 33101, USA.
Jaakola
Veli-Pekka
VP
Reusch
Regina
R
Valentine
Helene H
HH
Heikinheimo
Pirkko
P
Levay
Agata
A
Potter
Lincoln T
LT
Heimo
Heikki
H
Goldman
Adrian
A
Turner
George J
GJ
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001436:Bacteriorhodopsins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D015870:Gene Expression; D019617:Halobacterium salinarum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011941:Receptors, Adrenergic / Q000096:biosynthesis / Q000235:genetics*; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics; D011976:Receptors, Muscarinic / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013997:Time Factors
C547771:ADRA2B protein, human; D012333:RNA, Messenger; D011941:Receptors, Adrenergic; D018341:Receptors, Adrenergic, alpha-2; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D001436:Bacteriorhodopsins; C017827:bacterio-opsin