Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.

Anti-Tick Microbiota Vaccine Impacts Ixodes ricinus Performance during Feeding

The tick microbiota is a highly complex ensemble of interacting microorganisms. Keystone taxa, with a central role in the microbial networks, support the stability and fitness of the microbial communities. The keystoneness of taxa in the tick microbiota can be inferred from microbial co-occurrence networks. Microbes with high centrality indexes are highly connected with other taxa of the microbiota and are expected to provide important resources to the microbial community and/or the tick. We reasoned that disturbance of vector microbiota by removal of ubiquitous and abundant keystone bacteria may disrupt the tick-microbiota homeostasis causing harm to the tick host. These observations and reasoning prompted us to test the hypothesis that antibodies targeting keystone bacteria may harm the ticks during feeding on immunized hosts. To this aim, in silico analyses were conducted to identify keystone bacteria in the microbiota of Ixodes nymphs. The family Enterobacteriaceae was among the top keystone taxa identified in Ixodes microbiota. Immunization of α-1,3-galactosyltransferase-deficient-C57BL/6 (α1,3GT KO) mice with a live vaccine containing the Enterobacteriaceae bacterium Escherichia coli strain BL21 revealed that the production of anti-E. coli and anti-α-Gal IgM and IgG was associated with high mortality of I. ricinus nymphs during feeding. However, this effect was absent in two different strains of wild type mice, BALB/c and C57BL/6. This result concurred with a wide distribution of α-1,3-galactosyltransferase genes, and possibly α-Gal, in Enterobacteriaceae and other bacteria of tick microbiota. Interestingly, the weight of I. ricinus nymphs that fed on E. coli-immunized C57BL/6 was significantly higher than the weight of ticks that fed on C57BL/6 immunized with a mock vaccine. Our results suggest that anti-tick microbiota vaccines are a promising tool for the experimental manipulation of vector microbiota, and potentially the control of ticks and tick-borne pathogens.

Deciphering the transcriptional changes in Escherichia coli strains C41(DE3) and C43(DE3) that makes them a superior choice for membrane protein production.

Background: The production of membrane proteins for functional and structural protein analysis remains a bottleneck in the continuing quest for understanding biological systems. For recombinant membrane proteins, the Walker strains C41(DE3) and C43(DE3) are a valuable tool because they are capable of producing levels of functional protein that would otherwise be toxic to the cell. At the genome level, amongst only a handful of genetic changes, mutations in the lac UV5 promoter region upstream from the bacteriophage T7 RNA polymerase gene distinguish these strains from BL21(DE3) but do not inform on how the strains have adapted for superior production of recombinant membrane proteins. Results: Comparative transcriptomic analyses revealed a moderate change in gene expression in C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) under standard growth conditions. However, under the conditions used for membrane protein production (with plasmid carriage and addition of IPTG), the differential response of C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) was striking. Over 2000 genes were differentially expressed in C41(DE3) with a two-fold change and false discover rate < 0.01 and 1700 genes differentially expressed in C43(DE3) compared to their parent strain BL21(DE3). Conclusion : These results illuminate the cellular adaptations occurring in the Walker strains in response to minimal genetic changes. These changes in the transcriptome may help alleviate the toxic effects that can occur and improve membrane protein production. The BL21(DE3) derivatives strains C41(DE3) and C43(DE3), are adept to the process of membrane biogenesis in E. coli , making them superior to their parent strain for the production of membrane proteins and potentially other toxic proteins.

Deciphering the transcriptional changes in Escherichia coli strains C41(DE3) and C43(DE3) that makes them a superior choice for membrane protein production.

Background: The production of membrane proteins for functional and structural protein analysis remains a bottleneck in the continuing quest for understanding biological systems. For recombinant membrane proteins, the Walker strains C41(DE3) and C43(DE3) are a valuable tool because they are capable of producing levels of functional protein that would otherwise be toxic to the cell. At the genome level, amongst only a handful of genetic changes, mutations in the lacUV5 promoter region upstream from the bacteriophage T7 RNA polymerase gene distinguish these strains from BL21(DE3) but do not inform on how the strains have adapted for superior production of recombinant membrane proteins. Results: Comparative transcriptomic analyses revealed a moderate change in gene expression in C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) under standard growth conditions. However, under the conditions used for membrane protein production (with plasmid carriage and addition of IPTG), the differential response of C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) was striking. Over 2000 genes were differentially expressed in C41(DE3) with a two-fold change and false discover rate < 0.01 and 1700 genes differentially expressed in C43(DE3) compared to their parent strain BL21(DE3). Conclusion: These results illuminate the cellular adaptations occurring in the Walker strains to alleviate the toxic effects that can occur during membrane protein production, whilst providing changes in metabolism pathways required for membrane protein biogenesis. The BL21(DE3) derivatives strains C41(DE3) and C43(DE3), are adept to the process of membrane biogenesis in E. coli, making them superior to their parent strain for the production of membrane proteins and potentially other toxic proteins.

Deciphering the Transcriptional Changes in Escherichia Coli Strains C41(DE3) and C43(DE3) That Makes Them A Superior Choice for Membrane Protein Production.

Background: The overproduction of membrane proteins for functional and structural protein analysis remains a bottleneck in the continuing quest for understanding biological systems. For recombinant membrane proteins, the Walker strains C41(DE3) and C43(DE3) are a valuable tool because they are capable of producing levels of functional protein that would otherwise be toxic to the cell. At the genome level, amongst only a handful of genetic changes, mutations in the lac UV5 promoter region upstream from the bacteriophage T7 RNA polymerase gene distinguish these strains from BL21(DE3) but do not inform on how the strains have adapted for superior production of recombinant membrane proteins. Results: Comparative transcriptomic analyses revealed a moderate change in gene expression in C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) under standard growth conditions. However, under the conditions used for membrane protein production (with plasmid carriage and addition of IPTG), the differential response of C41(DE3) and C43(DE3) compared to their parent strain BL21(DE3) was striking. Over 2000 genes were differentially expressed in C41(DE3) with a two-fold change and false discover rate < 0.01 and 1700 genes differentially expressed in C43(DE3) compared to their parent strain BL21(DE3). Conclusion : These results illuminate the cellular adaptations occurring in the Walker strains to alleviate the toxic effects that can occur during membrane protein production whilst providing changes in metabolism pathways required for membrane protein biogenesis. The BL21(DE3) derivatives strains C41(DE3) and C43(DE3), are adept to the process of membrane biogenesis in E. coli , making them superior to their parent strain for the expression of membrane proteins and potentially other toxic proteins.

Expression of a novel gene encoding protease inhibitor from metagenome of sponge in Vietnam

Marine sponge is known as a “gold mine” of natural products from marine environment. Many novel bioactive compounds have been isolated from marine sponges and sponge-associated microorganisms such as antibiotics, anti-cancer compounds, protease inhibitors, etc. In this study, we selected a gene encoding protease inhibitor from metagenome of a sponge collected in Da Nang to express in Escherichia coli BL21(DE3). The gene PI-DN9 encoding protease inhibitor (1.3 kb) was cut off cloning vector pUC57/PI-DN9 containing gene PI-DN9 and inserted into expression vector pET32a(+), the recombinant vector pET32a(+)/PI-DN9 then was transformed and expressed in the E. coli strain BL21(DE3). Results showed that recombinant protein (50 kDa) was expressed successfully at 25°C, 1 mM of IPTG in 5 hours. The recombinant protein was purified using Ni-NTA affinity chromatography column. Western blot assay and bioactive assay showed good activity of the purified protein.

Ubiquitin-Specific Proteinase of E. coli Does Not Require Obligatory Presence of Dipeptide GlyGly at Processing Site

It was shown that unlike eukaryotic ubiquitin-specific proteinases (DUBs deubiquitinating enzymes) deubiquitinase ElaD E.coli does not need obligatory presence of GlyGly dipeptide at the processing site and is capable to process sites GlyGly and GlyAla with equal efficiency. At the same time similar to eukaryotic DUB the bacterial enzyme does not process sites with dipeptides AlaGly, AlaAla or GlyPro. Considering functional mission of the deubiquitinating agent of E. coli, decrease in its specificity can be considered as valuable evolutional acquisition allowing to expand a list of the molecular targets being attacked during pathogenesis. ubiquitin, DUB, E. coli proteinase, elaD, virulence factor The authors sincerely thank the staff of the NRC «Kurchatov Institute» -GosNIIgenetika Drs. Skorokhodova A. Yu. and Goulevitch A.Yu. for designing and supplying the strain BL21(DE3)Ae/aD for the present work.