scholarly journals Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1448
Author(s):  
Emiko Kinoshita-Kikuta ◽  
Eiji Kinoshita ◽  
Misaki Suga ◽  
Mana Higashida ◽  
Yuka Yamane ◽  
...  

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

2011 ◽  
Vol 34 (2) ◽  
pp. 359-364 ◽  
Author(s):  
Haiqin Chen ◽  
Xie Yan ◽  
Fengwei Tian ◽  
Yuanda Song ◽  
Yong Q. Chen ◽  
...  

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Yaroslav Chushak ◽  
Svetlana Harbaugh ◽  
Kathryn Zimlich ◽  
Bryan Alfred ◽  
Jorge Chávez ◽  
...  

2020 ◽  
Vol 20 ◽  
pp. 04004
Author(s):  
Ahmad Pandu Satria Wiratama ◽  
Aris Haryanto

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..


2016 ◽  
Vol 6 (1) ◽  
Author(s):  
L. Briand ◽  
G. Marcion ◽  
A. Kriznik ◽  
J. M. Heydel ◽  
Y. Artur ◽  
...  

1998 ◽  
Vol 64 (5) ◽  
pp. 1589-1593 ◽  
Author(s):  
Michael J. Weickert ◽  
Izydor Apostol

ABSTRACT Coexpression of di-α-globin and β-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed ≤0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of ≤0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to ∼20% of the level of E. colisoluble proteins also resulted in equivalent translational fidelity.


2015 ◽  
Vol 100 (1) ◽  
pp. 255-262 ◽  
Author(s):  
Keith W. Y. Kwong ◽  
Alan K. L. Ng ◽  
W. K. R. Wong

2013 ◽  
Vol 79 (12) ◽  
pp. 3802-3812 ◽  
Author(s):  
Juergen Mairhofer ◽  
Theresa Scharl ◽  
Karoline Marisch ◽  
Monika Cserjan-Puschmann ◽  
Gerald Striedner

ABSTRACTPlasmid-basedEscherichia coliBL21(DE3) expression systems are extensively used for the production of recombinant proteins. However, the combination of a high gene dosage with strong promoters exerts extremely stressful conditions on producing cells, resulting in a multitude of protective reactions and malfunctions in the host cell with a strong impact on yield and quality of the product. Here, we provide in-depth characterization of plasmid-based perturbations in recombinant protein production. A plasmid-free T7 system with a single copy of the gene of interest (GOI) integrated into the genome was used as a reference. Transcriptomics in combination with a variety of process analytics were used to characterize and compare a plasmid-free T7-based expression system to a conventional pET-plasmid-based expression system, with both expressing human superoxide dismutase in fed-batch cultivations. The plasmid-free system showed a moderate stress response on the transcriptional level, with only minor effects on cell growth. In contrast to this finding, comprehensive changes on the transcriptome level were observed in the plasmid-based expression system and cell growth was heavily impaired by recombinant gene expression. Additionally, we found that the T7 terminator is not a sufficient termination signal. Overall, this work reveals that the major metabolic burden in plasmid-based systems is caused at the level of transcription as a result of overtranscription of the multicopy product gene and transcriptional read-through of T7 RNA polymerase. We therefore conclude that the presence of high levels of extrinsic mRNAs, competing for the limited number of ribosomes, leads to the significantly reduced translation of intrinsic mRNAs.


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