Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

2000 ◽  
Vol 53 (4) ◽  
pp. 420-429 ◽  
Author(s):  
T. Clemente ◽  
D. Shah ◽  
M. Tran ◽  
D. Stark ◽  
S. Padgette ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Heterologous Expression ◽  
Rhodospirillum Rubrum ◽  
Pha Synthase ◽  
Expression Systems ◽  
Heterologous Expression Systems

scholarly journals Heterologous Expression of a Membrane-Spanning Auxin Importer: Implications for Functional Analyses of Auxin Transporters

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
David John Carrier ◽  
Norliza Tendot Abu Bakar ◽  
Karen Lawler ◽  
James Matthew Dorrian ◽  
Ameena Haider ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Mammalian Cells ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
Functional Assay ◽  
Expression Systems ◽  
Heterologous Expression Systems ◽  
Membrane Spanning ◽  
High Level

Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

Kv3.1–Kv3.2 Channels Underlie a High-Voltage–Activating Component of the Delayed Rectifier K+ Current in Projecting Neurons From the Globus Pallidus

1999 ◽  
Vol 82 (3) ◽  
pp. 1512-1528 ◽  
Author(s):  
R. Hernández-Pineda ◽  
A. Chow ◽  
Y. Amarillo ◽  
H. Moreno ◽  
M. Saganich ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Globus Pallidus ◽  
High Frequency ◽  
Calcium Binding ◽  
Repetitive Firing ◽  
Delayed Rectifier ◽  
Expression Systems ◽  
Electrophysiological Properties ◽  
Channel Proteins ◽  
Heterologous Expression Systems

The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3.1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than −10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1–Kv3.2 voltage-gated K+channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.

Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase

1996 ◽  
Vol 271 (3) ◽  
pp. C825-C832 ◽  
Author(s):  
T. A. Pressley ◽  
J. C. Allen ◽  
C. H. Clarke ◽  
T. Odebunmi ◽  
S. C. Higham
Keyword(s):  
Insect Cells ◽  
In Vitro Translation ◽  
Expression Systems ◽  
Amino Terminal ◽  
Cleavage Process ◽  
Heterologous Expression Systems ◽  
Terminal Structure ◽  
A Site

The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.

scholarly journals Heterodimerization of two pore domain K+ channel TASK1 and TALK2 in living heterologous expression systems

PLoS ONE ◽  
2017 ◽  
Vol 12 (10) ◽  
pp. e0186252 ◽  
Author(s):  
Yoshiaki Suzuki ◽  
Kanako Tsutsumi ◽  
Tatsuya Miyamoto ◽  
Hisao Yamamura ◽  
Yuji Imaizumi
Keyword(s):  
Heterologous Expression ◽  
K Channel ◽  
Expression Systems ◽  
Heterologous Expression Systems ◽  
Pore Domain

L-Type CaV1.2 Calcium Channels: From In Vitro Findings to In Vivo Function

2014 ◽  
Vol 94 (1) ◽  
pp. 303-326 ◽  
Author(s):  
Franz Hofmann ◽  
Veit Flockerzi ◽  
Sabine Kahl ◽  
Jörg W. Wegener
Keyword(s):  
Calcium Channel ◽  
Splice Variants ◽  
Kinetic Properties ◽  
Expression Systems ◽  
Smooth Muscle Contractility ◽  
Dependent Inactivation ◽  
Heterologous Expression Systems ◽  
Channel Properties

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca2+ itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that β-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVβ2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca2+-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

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