By M. Cristina Vega
This booklet offers complex expression applied sciences for the construction of protein complexes. due to the fact complexes lie on the center of recent biology, the expression, purification, and characterization of enormous quantities of top quality protein complexes is important for the fields of biomedicine, biotechnology, and structural biology. From co-expression in E. coli, yeast, mammalian and bug cells to advanced reconstitution from person subunits, this e-book bargains precious insights and assistance for profitable protein expressionists.
Across a number of sections readers will notice present possibilities for the creation of protein complexes in bacterial structures (including membrane proteins and cell-free co-expression), methylotrophic and non-methylotrophic yeasts, protozoa (Leishmania terantolae and Dictyostelium discoideum), baculovirus-infected insect cells, mammalian cells, crops and algae. advanced reconstitution from separately purified subunits or subcomplexes is mentioned as a complementary process. a final part introduces in brief many of the biophysical and structural characterization innovations for macromolecular complexes utilizing state of the art resolution scattering and nuclear magnetic resonance.
This paintings is a guided journey over probably the most robust and winning protein expression applied sciences, with a spotlight on co-expression and high-throughput purposes. it truly is addressed to every body drawn to the construction and characterization of macromolecular complexes, from college scholars who wish an obtainable description of the foremost co-expression structures to researchers in biomedicine and the lifestyles sciences looking for an up to date survey of accessible technologies.
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This booklet provides complicated expression applied sciences for the construction of protein complexes. seeing that complexes lie on the center of contemporary biology, the expression, purification, and characterization of enormous quantities of fine quality protein complexes is important for the fields of biomedicine, biotechnology, and structural biology.
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Extra resources for Advanced Technologies for Protein Complex Production and Characterization
AD stands for Acceptor-Donor fusion. ADD stands for Acceptor-Donor-Donor fusion. Not all possible fusion products are shown for clarity. Levels of multiresistance for product selection are indicated (top, right). All reactions occur in a single Eppendorf tube. Fusion products co-exist with educts. Productive fusion products are selected using (multi)antibiotic challenge, for example on a 96-well micro-titer plate (bottom). Desired Acceptor-Donor fusions are identified according to their resistance marker ‘bar-code’.
These properties, combined with the eukaryotic nature of yeasts, which allow them to perform many PTMs and to provide sophisticated folding machinery for the efﬁcient production of eukaryotic protein machines, have spurred the idea that yeasts should be used more frequently as the ﬁrst alternative microorganism to E. coli . For eukaryotic proteins of fungal origin, some naturally located in the cytoplasm (lactase, lipases) or secreted to the extracellular medium (glycosidases, peptidases), unicellular fungi (yeasts) or mycelial fungi (ﬁlamentous fungi) might be more appropriate hosts for expression, especially given the more efﬁcient secretory pathways of yeasts and fungi when compared with prokaryotic hosts.
Structure 11(11):1359–1367 3. Cai M, Huang Y, Sakaguchi K, Clore GM, Gronenborn AM, Craigie R (1998) An efﬁcient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J Biomol NMR 11(1):97–102 4. Hochkoeppler A (2013) Expanding the landscape of recombinant protein production in Escherichia coli. Biotechnol Lett 35(12):1971–1981 5. Bieniossek C, Nie Y, Frey D, Olieric N, Schafﬁtzel C, Collinson I, Romier C, Berger P, Richmond TJ, Steinmetz MO, Berger I (2009) Automated unrestricted multigene recombineering for multiprotein complex production.
Advanced Technologies for Protein Complex Production and Characterization by M. Cristina Vega