By M. Cristina Vega
This ebook offers complex expression applied sciences for the creation of protein complexes. in view that complexes lie on the center of recent biology, the expression, purification, and characterization of enormous quantities of top of the range protein complexes is essential for the fields of biomedicine, biotechnology, and structural biology. From co-expression in E. coli, yeast, mammalian and bug cells to complicated reconstitution from person subunits, this e-book bargains beneficial insights and counsel for profitable protein expressionists.
Across a number of sections readers will notice current possibilities for the creation of protein complexes in bacterial platforms (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 approach. a final part introduces in brief many of the biophysical and structural characterization recommendations for macromolecular complexes utilizing state of the art answer scattering and nuclear magnetic resonance.
This paintings is a guided travel over the most robust and winning protein expression applied sciences, with a spotlight on co-expression and high-throughput functions. it's addressed to all people attracted to the creation and characterization of macromolecular complexes, from college scholars who wish an available description of the most important co-expression structures to researchers in biomedicine and the existence sciences looking for an updated survey of obtainable technologies.
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Additional info for Advanced Technologies for Protein Complex Production and Characterization
An attractive feature of L. tarentolae is that the expression cassettes can be inserted into its genome with ease, yielding cell lines that can overexpress several protein chains simultaneously. Dictyostelium discoideum (discussed in Chap. 11 with ﬁlamentous fungi) has excelled for the production of cytoskeletal proteins, perhaps owing to the specializations of D. discoideum for a highly active, motile lifestyle. The social amoeba has the enzymatic machinery to decorate glycoproteins with nearly mammalian glycosylation patterns, a property that becomes interesting for pharmacological target proteins as an alternative to highly engineered mammalian glycosylation mimicking systems.
Many strains and plasmids are available to introduce several PTMs, to enable the secretion of expressed proteins (especially when they are small), to assist the folding of disulﬁde-bridge containing proteins, and to assemble coexpression constructs. Chapter 5 describes E. coli as a host for the overexpression of membrane proteins and Chap. 6 discusses the use of cell-free extracts for protein expression. B. megaterium (Chap. 7) is a better secretor than E. coli and has been used for the production of virulence factors.
Recombinant production offers solutions to these impediments, and a wide range of expression systems is available to produce proteins recombinantly in prokaryotic and eukaryotic hosts [31, 33–45]. Recombinant expression systems share in common that one or several DNA Y. Nie et al. 30 segments encoding for proteins, protein domains or multicomponent protein complexes are typically combined with DNA elements including DNAs that control transcription (promoters, terminators, others) and translation (ribosome binding sites, Shine-Dalgarno sequences, Kozak consensus sequences, enhancers, others) and inserted into a functional DNA module (plasmid, cosmid, artificial chromosome, genome, others).
Advanced Technologies for Protein Complex Production and Characterization by M. Cristina Vega