scholarly journals Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

2018 ◽  
Author(s):  
Rachel J. Harding ◽  
Peter Loppnau ◽  
Suzanne Ackloo ◽  
Alexander Lemak ◽  
Ashley Hutchinson ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Eukaryotic Expression ◽  
Critical Threshold ◽  
Expression Systems ◽  
Complex Samples ◽  
X Ray Scattering ◽  
Cag Expansion ◽  
Structural Work ◽  
Expansion Mutation ◽  
Eukaryotic Expression Systems

ABSTRACTThe gene mutated in Huntington’s disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.

scholarly journals Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells

Biochemistry ◽  
2020 ◽  
Vol 59 (39) ◽  
pp. 3757-3771
Author(s):  
Asma S. Al-Amoodi ◽  
Kosuke Sakashita ◽  
Amal J. Ali ◽  
Ruoyu Zhou ◽  
Jae Man Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Eukaryotic Expression ◽  
Expression Systems ◽  
Selectin Binding ◽  
Eukaryotic Expression Systems

scholarly journals Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity

1996 ◽  
Vol 320 (1) ◽  
pp. 129-135 ◽  
Author(s):  
Nathalie LAMARCHE ◽  
Gilles MATTON ◽  
Bernard MASSIE ◽  
Marc FONTECAVE ◽  
Mohamed ATTA ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ribonucleotide Reductase ◽  
Binding Capacity ◽  
Eukaryotic Expression ◽  
Eukaryotic Cells ◽  
Expression Systems ◽  
Simplex Virus ◽  
R2 Subunit ◽  
Eukaryotic Expression Systems

The R2 subunit of ribonucleotide reductase from herpes simplex virus type 2 was overproduced with prokaryotic and eukaryotic expression systems. The recombinant R2 purified by a two-step procedure exhibited a 3-fold higher activity when produced in eukaryotic cells. Precise quantification of the R2 concentration at each step of the purification indicated that the activity was not altered during the purification procedure. Moreover, we have observed that the level of R2 expression, in eukaryotic cells as well as in prokaryotic cells, did not influence R2 activity. Extensive characterization of the recombinant R2 purified from eukaryotic and prokaryotic expression systems has shown that both types of pure R2 preparations were similar in their 76 kDa dimer contents (more than 95%) and in their ability to bind the R1 subunit. However, we have found that the higher activity of R2 produced in eukaryotic cells is more probably related to a higher capability of binding the iron cofactor as well as a 3-fold greater ability to generate the tyrosyl free radical.

Eukaryotic Expression Systems: A Comparison

1996 ◽  
Vol 8 (3) ◽  
pp. 271-282 ◽  
Author(s):  
Sabine Geisse ◽  
Hermann Gram ◽  
Beate Kleuser ◽  
Hans P. Kocher
Keyword(s):  
Eukaryotic Expression ◽  
Expression Systems ◽  
Eukaryotic Expression Systems

scholarly journals A Brief Reminder of Systems of Production and Chromatography-Based Recovery of Recombinant Protein Biopharmaceuticals

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
B. Owczarek ◽  
A. Gerszberg ◽  
K. Hnatuszko-Konka
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Eukaryotic Expression ◽  
Expression Systems ◽  
Expression And Purification ◽  
Advantages And Disadvantages ◽  
Protein Molecules ◽  
The Face ◽  
Mature Field ◽  
Eukaryotic Expression Systems

Recombinant proteins are produced for various applications in laboratory and industrial settings. Among them, therapeutic applications have evolved into a mature field in recent years, affecting the face of contemporary medical treatment. This, in turn, has stimulated an ever-greater need for innovative technologies for the description, expression, and purification of recombinant protein biopharmaceuticals. Therefore, many biopharmaceuticals are synthesized in heterologous systems to obtain satisfactory yields that cannot be provided by natural sources. As more than 35 years has passed since the first recombinant biopharmaceutical (human insulin) successfully completed clinical trials in humans, we provide a brief review of the available prokaryotic and eukaryotic expression systems, listing the advantages and disadvantages of their use. Some examples of therapeutic proteins expressed in heterologous hosts are also provided. Moreover, technologies for the universal extraction of protein molecules are mentioned here, as is the methodology of their purification.

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