Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris

Protein Expression and Purification
N G AbdulaevK D Ridge

Abstract

The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal. Quantitative-competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome. The expression level in this transformant corresponded to approximately 0.3 mg of opsin per liter of cell culture (A600 = 1.0). Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction. Similar to retinal opsin, P. pastoris-expressed opsin migrated as a single band of approximately 37 kDa on SDS-PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (approximately 4-15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (lambda max 500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment. Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to t...Continue Reading

References

Feb 14, 1992·Biochemical and Biophysical Research Communications·H J HuangT T Kuo
Oct 1, 1991·Proceedings of the National Academy of Sciences of the United States of America·C H SungJ Nathans
Apr 1, 1990·Protein Engineering·S A ZozulyaP R Badalov
Dec 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·D D OprianH G Khorana
Nov 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·H G KhoranaD A Thompson
May 21, 1974·Biochemistry·D S Papermaster, W J Dreyer
Jan 1, 1983·Biophysics of Structure and Mechanism·P A HargraveP Argos
Oct 28, 1980·Biochemistry·J K WangP A Hargrave
Jul 24, 1980·Biochimica Et Biophysica Acta·S TsunasawaH Shichi
Aug 1, 1995·Quarterly Reviews of Biophysics·R Grisshammer, C G Tate
Apr 11, 1995·Proceedings of the National Academy of Sciences of the United States of America·K D RidgeL L Yao
Aug 1, 1993·Bio/technology·J M CreggW C Raschke
Apr 26, 1994·Proceedings of the National Academy of Sciences of the United States of America·S KaushalH G Khorana
Apr 22, 1993·Nature·G F SchertlerR Henderson
Mar 29, 1996·The Journal of Biological Chemistry·K D RidgeN G Abdulaev
Oct 15, 1996·Proceedings of the National Academy of Sciences of the United States of America·R MollaaghababaH G Khorana
Nov 20, 1953·The Journal of General Physiology·G WALD, P K BROWN

❮ Previous
Next ❯

Citations

Feb 8, 2006·Applied and Environmental Microbiology·Mark J DanielsMark Yeager
Jun 13, 2002·Proceedings of the National Academy of Sciences of the United States of America·Oleg A SineshchekovJohn L Spudich
Oct 11, 2003·FEBS Letters·Douglas A GriffithDieter Oesterhelt
Aug 19, 2007·Journal of Structural Biology·Charles R Midgett, Dean R Madden
Nov 27, 2004·Journal of Molecular Recognition : JMR·Rachel Daly, Milton T W Hearn
Sep 10, 2003·Biochemical and Biophysical Research Communications·Sreelatha T ReddyNancy M Dahms
Feb 23, 2002·Protein Expression and Purification·Valérie SarramegnaFranck Talmont
Oct 26, 2000·Protein Expression and Purification·P Newton-VinsonD E Edmondson
Nov 21, 2007·Applied Biochemistry and Biotechnology·Pingzuo LiV Renugopalakrishnan
Jan 8, 2011·Journal of Molecular Modeling·Seisuke FujitaNorihisa Fujita
Jan 21, 2000·FEMS Microbiology Reviews·J L Cereghino, J M Cregg
Oct 30, 2018·Chemical Reviews·Oh Seok KwonJyongsik Jang

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.