scholarly journals An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hongmin Cai ◽  
Hebang Yao ◽  
Tingting Li ◽  
Cedric A. J. Hutter ◽  
Yanfang Li ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Expression ◽  
Fluorescent Proteins ◽  
High Sensitivity ◽  
Green Fluorescent Proteins ◽  
Affinity Tag ◽  
Membrane Protein Expression ◽  
Green Fluorescent ◽  
Membrane Protein Stability ◽  
Fluorescent Tag

AbstractGreen fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.

scholarly journals Coherent Dynamics of Photoexcited Green Fluorescent Proteins

2001 ◽  
Vol 86 (15) ◽  
pp. 3439-3442 ◽  
Author(s):  
Riccardo A. G. Cinelli ◽  
Valentina Tozzini ◽  
Vittorio Pellegrini ◽  
Fabio Beltram ◽  
Giulio Cerullo ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Coherent Dynamics ◽  
Green Fluorescent

Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

2011 ◽  
Vol 100 (9) ◽  
pp. 3939-3950 ◽  
Author(s):  
Katsuaki Ito ◽  
Yasuo Uchida ◽  
Sumio Ohtsuki ◽  
Sanshiro Aizawa ◽  
Hirotaka Kawakami ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Expression ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Cynomolgus Monkeys ◽  
Membrane Protein Expression ◽  
Blood Brain

scholarly journals Sex Hormones Regulate Rat Hepatic Monocarboxylate Transporter Expression and Membrane Trafficking

2017 ◽  
Vol 20 (1) ◽  
pp. 435 ◽  
Author(s):  
Jieyun Cao ◽  
Michael Ng ◽  
Melanie A Felmlee
Keyword(s):  
Sex Differences ◽  
Membrane Protein ◽  
Protein Expression ◽  
Estrous Cycle ◽  
Sex Hormones ◽  
Membrane Trafficking ◽  
Drug Disposition ◽  
Membrane Expression ◽  
Membrane Protein Expression ◽  
Estrous Cycle Stage

Purpose: Monocarboxylate transporters (MCTs) are involved in the transport of monocarboxylates such as ketone bodies, lactate, and pharmaceutical agents. CD147 functions as an ancillary protein for MCT1 and MCT4 for plasma membrane trafficking. Sex differences in MCT1 and MCT4 have been observed in muscle and reproductive tissues; however, there is a paucity of information on MCT sex differences in tissues involved in drug disposition. The objective of the present study was to quantify hepatic MCT1, MCT4 and CD147 mRNA, total cellular and membrane protein expression in males, over the estrous cycle in females and in ovariectomized (OVX) females. Method: Liver samples were collected from females at the four estrous cycle stages (proestrus, estrus, metestrus, diestrus), OVX females and male Sprague-Dawley rats (N = 3 – 5). Estrus cycle stage of females was determined by vaginal lavage. mRNA and protein (total and membrane) expression of MCT1, MCT4 and CD147 was evaluated by qPCR and western blot analysis. Results: MCT1 mRNA and membrane protein expression varied with estrous cycle stage, with OVX females having higher expression than males, indicating that female sex hormones may play a role in MCT1 regulation. MCT4 membrane expression varied with estrous cycle stage with expression significantly lower than males. MCT4 membrane expression in OVX females was also lower than males, suggesting that androgens play a role in membrane expression of MCT4. Males had higher membrane CD147 expression, whereas there was no difference in whole cell protein and mRNA levels suggesting that androgens are involved in regulating CD147 membrane localization. Conclusions: This study demonstrates hepatic expression and membrane localization of MCT1, MCT4 and CD147 are regulated by sex hormones. Sex differences in hepatic MCT expression may lead to altered drug disposition, so it is critical to elucidate the underlying mechanisms in the sex hormone-dependent regulation of MCT expression. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

scholarly journals Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

2017 ◽  
Vol 114 (11) ◽  
pp. E2146-E2155 ◽  
Author(s):  
Chi-Yun Lin ◽  
Johan Both ◽  
Keunbong Do ◽  
Steven G. Boxer
Keyword(s):  
Quantum Yield ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Point Mutations ◽  
Photoactive Yellow Protein ◽  
Green Fluorescent Proteins ◽  
Protein Protein Interactions ◽  
Low Efficiency ◽  
Green Fluorescent ◽  
Rate Limiting

Split GFPs have been widely applied for monitoring protein–protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics–function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

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