Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase

Vibrio natriegens is the fastest-growing bacteria, and its doubling time is less than 10 min. At present, the T7 expression system has been introduced into V. natriegens for heterologous protein expression, including the commercial strain Vmax1 and the variant VnDX,2 which is a backup expression chassis of Escherichia coli BL21­(DE3). However, the strength of the existing T7 expression system is not optimal for every recombinant protein. The different expression strengths of T7 RNA polymerase (T7 RNAP) can be obtained by changing the promoter and ribosome binding site (RBS) sequences of T7 RNAP at different transcription and translation levels. In this work, we obtained a robust VnDX variant library with the fine-tuning T7 RNAP using the industrially used enzyme glucose dehydrogenase (GDH) as the reporter protein. Among this library, the variant VnDX-tet, whose promoter of T7 RNAP was changed from PlacUV5 to Ptet, showed that the reporter enzyme GDH activity was increased by 109% by the T7 expression system. Similarly, variants with different T7 RNAP translation levels were obtained by changing RBS sequences upstream of T7 RNAP, and the results showed that the variant VnDX-RBS12/pGDH had the highest GDH activity, which increased by 12.6%. The VnDX variant library constructed in this study with different T7 expression strengths provides a choice for expressing various recombinant proteins, greatly expanding the application of V. natriegens

GPR108 suppresses signaling through modulating MyD88 during stimulation.

(A) The enhanced MyD88 signals in primary Gpr108-/- macrophages compared to that in Gpr108+/+ macrophages when stimulated with LPS (100 ng/ml), Poly (I:C) (5 μg/ml), R484 (10 μg/ml) and CPG (1μM) at different time points. (B) Immunoblotting analysis of MyD88 in immortalized macrophages transduced with vector control or GPR108 cDNA tagged with cherry after LPS stimulation at different time points. (C) HEK293 cells were co-transfected with Flag-MyD88 and HA-vector or HA-tagged GPR108. MyD88 and GPR108 were measured by immunoblotting with anti-HA and anti-Flag antibodies. (D) HEK293 cells were co-transfected with Flag-TLR4 and HA-vector or GPR108 or GPR108 mutant m1, m2 and m3. Endogenous MyD88 were measured by immunoblotting after stimulation with LPS at different time points. All experiments were repeated for at least three times, and the data were the representative of similar results. Right lane listed the molecular size in kilodaltons (kD).</p

Generation of <i>Gpr108</i>-null mice.

(A) Schematic diagram of construction of Gpr108 knockout mice. The murine Gpr108 locus contains 17 exons. Using a targeting BAC bearing the selection marker tk-2A-neo flanked by R4 integrase attB and attP sites, exons 1 to 17 were deleted, yielding the MT1 ES cell line. After expression of R4 integrase in MT1 cells, the resistance elements were removed as shown in MT2. The probes used for MLPA, 5wt, 3wt, 5m, 3m, neo, C1 and C2 are indicated. The primers P1/P2, P3/P4 used for PCR genotyping and P5/P6 for R4 deletion test are also shown. (B) Representative MLPA fragment analysis of 5wt, 3wt, 5m, 3m, neo, C1, C2 for different target events in wild-type cells as well as MT1 and MT2 targeted ES cells. Using HP1 (C1) and ITGB3 (C2) as internal controls to normalize intensities, the 5wt and 3wt signal intensities were observed to be diminished by approximately one half in correctly targeted MT1 cells compared to those in wt cells and the copy numbers of 5m, 3m and neo were identified as single copy or greater by analysis of all ES clones exhibiting resistance to G-418. In MT2 ES cells, the neo MLPA signal was not observed following excision by R4 integrase. (C) Mouse genotypes by PCR of wild-type (WT), heterozygous (Het) and homozygous (KO) mice are indicated. (D) Gpr108 mRNA abundance in WT, Het and KO MEF cells. Gpr108 mRNA was absent from KO MEF cells and present in Het at approximately 50% of the level found in wild-type MEF cells. (E) Gpr108 mRNA detection in spleen and lung of WT and KO mice. Gpr108 mRNA was absent in tissues from KO mice. (F). mRNA transcriptome panel of immune response and immediate early genes (IEGs) in BMDM cells derived from Gpr108+/+ (n = 3) and Gpr108-/- (n = 3) mice in the absence or presence of different TLR agonists. Each column presents the mRNA expression of BMDM cells without or with different treatments. The intensity represents the magnitude of the difference. Red and green denote low and high expression, respectively.</p

GPR108 interacted with TLRs and their partners.

(A) Confocal microscopy analysis of colocalization of GPR108 and TLRs in Hela cells transfected with mCherry-GPR108 (red) with CFP-TLR3 or EGFP-TLR7 (green), or EGFP-GPR108 (green) with mCherry-TLR4 or TLR9 (red). Nuclei were stained by Hochest (blue). Scale bars, 10μm. (B) Flag or HA tagged GPR108 was co-transfected with HA or Flag tagged TLRs (TLR3, TLR4 and TLR7) in HEK293 cells. Flag tagged proteins were immunoprecipitated with M2 anti-flag beads. Both whole cell lysates (WCL) and immunoprecipitates (IP) samples were subjected for immunoblotting with anti-flag and anti-HA antibodies. (C) Flag or HA tagged GPR108 was co-transfected with HA tagged or Flag tagged adaptor proteins (TIRAP and MyD88). Flag or HA tagged TLR4 was used as positive control and LRSAM as negative control. Cells were immunoprecipitated with M2 beads and both whole cell lysates and IP samples were subjected for blotting with anti-Flag and anti-HA antibodies. (D) Flag-MyD88 and HA-GPR108 were co-transfected with or without HA-TLR4 in HEK293 cells. Cells were immunoprecipitated with M2 beads and both whole cell lysates and IP samples were subjected for blotting with anti-Flag and anti-HA antibodies. All experiments were repeated for at least three times, and the data were the representative of similar results. Left margin listed the molecular size in kilodaltons (kD).</p

Solubility of l‑Phosphinothricin and Its Hydrochloride in Water + (Methanol and Ethanol) Binary Solvent Mixtures from 278.15 to 318.15 K

The solubility of l-phosphinothricin (l-PPT) and its hydrochloride in water + (methanol and ethanol) binary solvent mixtures was experimentally measured from 278.15 to 318.15 K by a static analytic method under atmospheric pressure. The experimental data showed that the solubility increased with the increasing temperature and decreased with the increase of alcohol concentration in binary solvent mixtures. The experimental data were correlated by the modified Apelblat equation and combined nearly ideal binary solvent/Redlich–Kister model. The results indicated that the modified Apelblat equation showed better agreement with the experimental data. The parameters characterizing preferential solvation of l-PPT in water + methanol binary solvents had been calculated. The results showed that the preferential solvation of l-PPT was related to the solvent composition and temperature

GPR108 reduces MyD88 binding on TLR4 which might be attributable to its ability of blocking MyD88 ubiquitination through negatively regulating E3 ligase TRAF6.

(A) HEK293T cells were co-transfected with Flag-TLR4 and HA-MyD88 with or without GPR108. Cells stimulated with LPS at different time points were subjected for immunoprecipitated with M2 anti-flag beads. IP samples were blotted with anti-MyD88 and anti-flag antibodies and whole cell lysates were blotted with anti-MyD88. (B) HEK293T cells were transfected with Flag-GPR108 as well as HA-TRAF6 or HA-vector. Cells were immunoprecipitated and subjected for blotting with anti-HA and anti-Flag antibodies. (C) HEK293T cells were co-transfected with Flag-MyD88 and Myc-Ubiquitin as well as HA vector or HA-GPR108. Cells were subjected for IP and immunoblotting analysis for anti-Myc, anti-Flag and anti-HA antibodies. (D) HEK293 cells were transfected with HA-vector or GPR108-HA. Cells were subjected for blotting with anti-HA, TRAF6 and actin antibodies.</p

GPR108 was negatively regulated by TIRAP which feedback on TRAF6 in turn.

(A) Immunoblotting analysis of HEK293T cells transfected with HA tagged TIRAP, TIRAPP125H and GPR108 without flag-TLR4 or with flag-TLR4. Whole cell lysates were blotting with anti-HA antibody. (B) Immunoblotting analysis of HEK293T cells transfected with HA tagged TIRAP, TIRAPP125H and GPR108 with flag-TLR4. Whole cell lysates were blotted with antibodies anti-flag as well as anti-MyD88 with actin as an internal control. (C) HEK293T cells were transfected with HA-GPR108 and Flag vector or TIRAP at different DNA amount. Cell lysates were subjected for immunoblotting analysis against Flag, HA and TRAF6. All experiments were repeated for at least three times, and the data were the representative of similar results.</p

TLRs-triggered immune response was enhanced in <i>Gpr108</i>-null cells.

(A) Primary macrophage cells derived from litter mated Gp108+/+ and Gpr108-/- mice were treated with LPS (1 μg/ml, TLR4), Poly (I:C) (5 μg/ml, TLR3), immiquimod (50 μg/ml, TLR7) and R484 (10 μg/ml, TLR7). IL-6 and TNFα production were measured by ELISA after 18 hours treatment. The expression of IFNβ was determined by RT-qPCR after 8 hours treatment. (B) IL-6 and TNFα production were measured in immortalized Gpr108+/+ and Gpr108-/- macrophages treated with LPS (100 ng/ml) at different time points. (C) IL-6 and TNFα production were measured in primary Gpr108+/+ and Gpr108-/- MEF cells treated with LPS (100 ng/ml) for 18 hours. (D) iBMDM cells were transduced with a lenti-virus NF-κB reporter containing luciferase and treated with LPS (100 ng/ml) and immiquimod (50 μg/ml) for 18 hours. The luciferase activities were measured as fold increase by normalizing to the untreated cells as 1. (E) BMDM cells were stimulated with LPS (100 ng/ml), Poly (I:C) (5 μg/ml), R484 (10 μg/ml) and CPG (1μM) at different time points. The phosphorylated IRF3, IKBα and phosphorylated IKBα were measured by immunoblotting analysis with actin protein as its loading control. Data were shown as mean±SD (A-D). *p < 0.05, **p < 0.01, ***p<0.001. All experiments were repeated for at least three times.</p

GPR108 works as a dual functional regulator to balance the TLR-triggered immune response.

For active immune response to defend the invading pathogens, TRAF6 mediates K63-linked MyD88 ubiquitination conferring its higher binding affinity to TLR4-TIRAP and inducing the downstream signaling activation through MyD88-dependent pathway (right side). At the same time, GPR108 expression is going up upon stimulation. It may mediate some unknown activation pathway in synergy with the TLR-triggered signaling. However, when signaling goes up higher, the elevated GPR108 would damp TRAF6 and reduce MyD88’s ubiquitination level which attenuates MyD88’s binding to TLR4 and collapses downstream signaling. So GPR108 suppresses the signaling by binding the de-ubiquitinated MyD88 that might possess higher affinity to GPR108 than TLR4 (left side). This regulatory loop will keep the signal activation within the appropriate range. For another level of counter regulation, TIRAP could restrict GPR108 which might act as an activator through an unknown independent pathway.</p

GPR108 works as an activator but limits TLRs-triggered NF-κB and IFNβ response.

(A) Two Gpr108-inducible clones 1–1 and 2–2, derived from Gpr108-/- iBMDM cells were stimulated with Dox (1μg/ml) for 48 hours. The expressions of Gpr108 were determined by RT-qPCR with actin gene as an internal control. (B) Two Gpr108-inducible clones 1–1 and 2–2 were transduced with NF-κB reporter containing luciferase. Cells were pretreated with or without Dox for 48 hours and then the luciferase activities were measured after the stimulation with LPS (100 ng/ml) and immiquimod (50 μg/ml) for 18 hours. (C) Gene expressions of IL-1β, IL-6, TNFα and IFNβ in DOX treated or untreated cells (clone1-1) (48 hours) were analyzed after exposure to LPS (100ng/ml) and immiquimod (50 μg/ml) for 1.5 hours. Actin was used as an internal control. (D) HEK293 cells were transfected with NF-κB-luciferase reporter and Flag-vector or Flag-TLRs, together with GFP or GPR108 plasmids. The NF-κB luciferase activities (fold increase) were analyzed after 48 hours transfection. (E) HEK293/TLR3, TLR4 and TLR7 stable cell lines were transfected with NF-κB-luciferase or IFNβ-luciferase reporter, together with control vector or Gpr108. Cells were treated with their corresponding agonists Poly (I:C) (TLR3), LPS (TLR4) and R484 (TLR7) at different doses for 18 hours and were analyzed for luciferase activities (fold increase). (F) Schematic diagram of the Gpr108 mutants: m1, an N-terminal domain deletion mutant with a leader peptide (green box); m2, loop3 replacement (AVPFQ (red line) was replaced with GGGGS (green line)); m3, a C-terminal domain deletion (purple line). (G) HEK293 cells were transfected with NF-κB-luciferase reporter, together with HA-vector, Gpr108 or Gpr108 mutants. The luciferase activities (fold increase) were analyzed after 48 hours transfection. Data were shown as mean±SD.*p < 0.05, **p < 0.01, ***p<0.001. All the experiments were repeated for at least three times.</p