New tools for recombinant protein production in Escherichia coli: A 5-year update.

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost-effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins.

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.

A pore-forming toxin enables Serratia a nonlytic egress from host cells.

Several pathogens co-opt host intracellular compartments to survive and replicate, and they thereafter disperse progeny to prosper in a new niche. Little is known about strategies displayed by Serratia marcescens to defeat immune responses and disseminate afterwards. Upon invasion of nonphagocytic cells, Serratia multiplies within autophagosome-like vacuoles. These Serratia-containing vacuoles (SeCV) circumvent progression into acidic/degradative compartments, avoiding elimination. In this work, we show that ShlA pore-forming toxin (PFT) commands Serratia escape from invaded cells. While ShlA-dependent, Ca2+ local increase was shown in SeCVs tight proximity, intracellular Ca2+ sequestration prevented Serratia exit. Accordingly, a Ca2+ surge rescued a ShlA-deficient strain exit capacity, demonstrating that Ca2+ mobilization is essential for egress. As opposed to wild-type-SeCV, the mutant strain-vacuole was wrapped by actin filaments, showing that ShlA expression rearranges host actin. Moreover, alteration of actin polymerization hindered wild-type Serratia escape, while increased intracellular Ca2+ reorganized the mutant strain-SeCV actin distribution, restoring wild-type-SeCV phenotype. Our results demonstrate that, by ShlA expression, Serratia triggers a Ca2+ signal that reshapes cytoskeleton dynamics and ends up pushing the SeCV load out of the cell, in an exocytic-like process. These results disclose that PFTs can be engaged in allowing bacteria to exit without compromising host cell integrity.

Calcineurin regulates progressive motility activation of Rhinella (Bufo) arenarum sperm through dephosphorylation of PKC substrates.

Animals with external fertilization, as amphibians, store their sperm in a quiescent state in the testis. When spermatozoa are released into natural fertilization media, the hypotonic shock triggers activation of sperm motility. Rhinella (Bufo) arenarum sperm are immotile in artificial seminal plasma (ASP, resembling testicular plasma tonicity) but acquire in situ flagellar beating upon dilution. However, if components from the egg shelly coat are added to this medium, motility shifts to a progressive pattern. Recently, we have shown that the signal transduction pathway required for in situ motility activation involves a rise in intracellular cAMP through a transmembrane adenylyl cyclase and activation of PKA, mostly in the midpiece and in the sperm head. In this report, we demonstrate that activation of calcineurin (aka PP2B and PPP3) is required for the shift from in situ to progressive sperm motility. The effect of calcineurin is manifested by dephosphorylation of PKC substrates, and can be promoted by intracellular calcium rise by Ca(2+) ionophore. Both phosphorylated PKC substrates and calcineurin localized to the flagella, indicating a clear differentiation between compartmentalization of PKA and calcineurin pathways. Moreover, no crosstalk is observed between these signaling events, even though both pathways are required for progressive motility acquisition as discussed.

ß-hexosaminidase from Xenopus laevis eggs and oocytes: from gene to immunochemical characterization.

Glycosidases are present both in sperm and eggs in vertebrates and have been associated with different fertilization steps as gamete binding, egg coat penetration, and polyspermy prevention. In this manuscript, we have analyzed the activity of different glycosidases of Xenopus laevis eggs. The main activity corresponded to N-acetyl-ß-D-glucosaminidase (Hex), which was reported to participate both in gamete binding and polyspermy prevention among phylogenetically distant animals. We have raised homologous antibodies against a recombinant N-terminal fragment of a X. laevis Hex, and characterized egg's Hex both by Western blot and immunohistochemical assays. Noteworthy, Hex was mainly localized to the cortex of animal hemisphere of full-grown oocytes and oviposited eggs, and remained unaltered after fertilization. Hex is constituted by different pair arrangements of two subunits (α and ß), giving rise to three possible Hex isoforms: A (αß), B (ßß), and S (αα). However, no information was available regarding molecular identity of Hex in amphibians. We present for the first time the primary sequences of two isoforms of X. laevis Hex. Interestingly, our results suggest that α- and ß-like subunits that constitute Hex isoforms could be synthesized from a same gene in Xenopus, by alternative exon use. This finding denotes an evolutionary divergence with mammals, where α and ß Hex subunits are synthesized from different genes on different chromosomes.