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Introduction: The Human papillomaviruses (HPV) main capsid protein L1 is naturally capable to self-assemble as virus-like particles (VLPs). There are different recombinant protein expression systems such as bacteria, yeast, insect, plant, and mammalian cells for generation of VLP-based candidate vaccines targeting various pathogens. In this study, we produced HPV-L1 protein by BL21/pET32a expression system and VLP production was confirmed.Material & Method: The recombinant plasmid pET32/L1 was transformed into Escherichia. coli BL21 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/L1 were assessed by restriction endonucleases HindIII and XhoI and sequence analysis. The expression of HPV16-L1 fusion protein in E. coli BL21was identified by SDS-PAGE and Western blotting. VLPs were evaluated using electron microscopy.Result: A codon-optimized L1 gene was expressed in BL21 under the control of the T7/lac promoter. Purification of L1 protein was achieved after Ni NTA chromatography. The 60kDa protein was detected in the lysates of BL21, recognized as HPV16- L1 protein by Western blotting. VLPs were confirmed using electron microscopy.Conclusion: In this study, we established a high-efficient recombinant E. coli expression system for the production of HPV 16- L1 protein. The generated L1 protein was correctly self-assembled into VLPs. Therefore, BL21/pET32a as a prokaryotic expression system is a potent tool for HPV16-L1 VLP production.

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.

Background: The human papillomavirus (HPV) main capsid protein L1 is naturally capable of self-assembly as virus-like particles (VLPs). There are different recombinant protein expression systems, such as bacteria, yeast, insect, plant, and mammalian cells, for the generation of VLP-based candidate vaccines targeting various pathogens. In this study, we produced the HPV16-L1 protein by BL21/pET32a expression system, and VLP production was confirmed. Methods: The recombinant plasmid pET32/L1 was transformed into Escherichia coli BL21 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/L1 were assessed by restriction endonucleases HindIII and XhoI and nested polymerase chain reaction (PCR). The expression of HPV16-L1 fusion protein in Escherichia coli BL21 was identified by SDS-PAGE and western blotting. Electron microscopy was used to evaluate VLP formation. Results: A codon-optimized L1 gene was expressed in BL21 under the control of the T7/lac promoter. Purification of L1 protein was achieved after Ni NTA chromatography. The 60 kDa protein was detected in the lysates of BL21, recognized as HPV16- L1 protein by western blotting. The VLPs were confirmed using electron microscopy. Conclusion: In this study, we established an efficient recombinant E. coli expression system for the production of HPV 16- L1 protein. The generated L1 protein was correctly self-assembled into VLPs. Therefore, BL21/pET32a as a prokaryotic expression system is a potent tool for HPV16-L1 VLP production. [ABSTRACT FROM AUTHOR]

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.

Background: Parathyroid Hormone (PTH) is secreted by parathyroid glands and controls the level of calcium in bones and kidney. PTH is a small polypeptide with 84 amino acids, but the first 34 amino acids of which are enough for hormone biological activity and can be used in the treatment of Osteoporosis. The expression efficiency of recombinant human parathyroid hormone rhPTH (1-34) or Teriparatide using a cleavable fusion protein strategy was compared in two strains of E. coli. Materials and Methods: A cassette was designed and fully synthesized for prokaryotic expression of rhPTH using pET system. From 5’ to 3’, the cassette consisted of: Trx tag to increase the solubility of protein, His tag for purification and detection of protein, enterokinase site to cleave all fusion moieties, and an optimized gene code for Teriparatide corresponding to the amino acid sequence of hPTH. This cassette was cloned into pET32a vector. The vector was simultaneously transformed and expressed in two different E. coli strains. The ability of strains for expression of this recombinant pharmaceutical was compared. Early expression was confirmed by SDS-PAGE and Western Blotting. The soluble fusion protein was harvested and purified by immobilized affinity chromatography. Then the fusion moiety was released from Teriparatide by enterokinase digestion. Results: The fusion form of rhPTH was efficiently expressed in both E. coli strains. However, the percentage of the target protein to the total protein content in Rosetta-gami was more than its amount in BL21 (­60 % vs 25%).The fusion protein was highly purified with Ni-NTA column. Up to 18.5 mg/ml of pure fusion protein has been obtained from 1-liter Rosetta-gami strain of E. coli. The pure Teriparatide was released by enterokinase digestion. Conclusion: The pure rhPTH (1-34) produced here, could be the subject for biological activity and quality control assessments, and following formulation processing, it could be applied as a peptide drug in the treatment of Osteoporosis.

DT-Diaphorase (DTD) belonging to the oxidoreductase family, is among the most important enzymes and is of great significance in present-day biotechnology. Also, it has potential applications in glucose and pyruvate biosensors. Another important role of the DTD enzyme is in the detection of Phenylketonuria disease. According to the above demands, at first, we tried to study molecular cloning and production of recombinant DTD in E. coli BL21 strain. We have successfully cloned, expressed, and purified functionally active diaphorase. The amount of enzyme was increased in 10-h using IPTG induction, and the recombinant protein was purified by Ni–NTA agarose affinity chromatography. After that, the kinetic and thermodynamic parameters of the enzyme, optimum temperature and pH were also investigated to find more in-depth information. In the end, to represent the connections between the structures and function of this enzyme, the molecular dynamics simulations have been considered at two temperatures in which DTD had maximum and minimum activity (310 and 293 K, respectively). The results of MD simulations indicated that the interaction between NADH with phenylalanine 232 residue at 310 K is more severe than other residues. So, to investigate the interaction details of NADH/PHE 232 the DFT calculations were done. [ABSTRACT FROM AUTHOR]

Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB), and/or the isocitrate lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110, and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate-forming background. Despite low 3HB titers in low-cell-density screening, 3HB-producing BL21 produced five times less acetic acid per mole of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L−1 h−1) and the highest 3HB concentration (16.3 g L−1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind. [ABSTRACT FROM AUTHOR]

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains RosettaTM(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain RosettaTM(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin. Keywords: C. perfringens beta toxin (CPB); expression; RosettaTM; BL21

Recombinant protein production is at the core of biotechnology and numerous molecular tools and bacterial strains have been developed to make the process more efficient. One commonly used generic solution is to supply extra copies of low-abundance tRNAs to compensate for the presence of complementary rare codons in genes-of-interest. Here we show that such extra tRNA, supplied by the commonly used pLysSRARE2 plasmid, can cause two side effects: (1) growth and gene expression can be impaired, and (2) apparent positive effects can be caused by differential expression of the lysozyme gene encoded on the same plasmid and not the tRNAs per se. These phenomena seem to have been largely overlooked despite the huge popularity of the T7/pET-based systems for bacterial protein production. [ABSTRACT FROM AUTHOR]

Acetate production is one of the most striking differences between Escherichia coli K12 and BL21 strains. Transcription of acetate metabolism genes is regulated. Additionally, acetyl-CoA synthetase, which activates acetate to acetyl-CoA, is regulated by post-translational acetylation. The aim of this study was to understand the contribution of reversible protein lysine acetylation to the regulation of acetate metabolism in E. coli BL21. The phenotypic differences between both strains were especially important in the presence of acetate. The high expression of acetyl-CoA synthetase ( acs) in glucose exponential phase in BL21 allows the simultaneous consumption of acetate and glucose. Lack of catabolite repression also affected its post-translational regulator, the protein acetyltransferase ( patZ). The effect of the deletion of cobB (encoding a sirtuin-like protein deacetylase) and patZ genes depended on the genetic background. The deletion of cobB in both strains increased acetate production and decreased growth rate in acetate cultures. The deletion of patZ in BL21 suppressed acetate overflow in glucose medium and increased the growth rate in acetate cultures. Differences on acetate overflow between BL21 and K12 strains are caused by many overlapping factors. Two major contributing effects were identified: (1) the expression of acs during exponential growth is not repressed in the BL21 strain due to concomitant cAMP production and (2) the acetyl-CoA synthetase activity is more tightly regulated by protein acetylation in BL21 than in the K12. Altogether these differences contribute to the lower acetate overflow and the improved ability of E. coli BL21 to consume this metabolite in the presence of glucose. [ABSTRACT FROM AUTHOR]

Deletion of both iclR and arcA in E. coli profoundly alters the central metabolic fluxes and decreases acetate excretion by 70%. In this study we investigate the metabolic consequences of both deletions in E. coli BL21 (DE3). No significant differences in biomass yields, acetate yields, CO yields and metabolic fluxes could be observed between the wild type strain E. coli BL21 (DE3) and the double-knockout strain E. coli BL21 (DE3) Δ arcAΔ iclR. This proves that arcA and iclR are poorly active in the BL21 wild type strain. Noteworthy, both strains co-assimilate glucose and acetate at high glucose concentrations (10-15 g l), while this was never observed in K12 strains. This implies that catabolite repression is less intense in BL21 strains compared to in E. coli K12. [ABSTRACT FROM AUTHOR]

Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with NHCl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective C-N isotopic-labeled nucleotides for synthesizing biologically important RNAs. [ABSTRACT FROM AUTHOR]

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K-12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6-deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by-product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host-specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli. [ABSTRACT FROM AUTHOR]

Escherichia coli strain BL21 is commonly used as a host strain for protein expression and purification. For structural analysis, proteins are frequently isotopically labeled with deuterium (2H), 13C, or 15N by growing E. coli cultures in a medium containing the appropriate isotope. When large quantities of fully deuterated proteins are required, E. coli is often grown in minimal media with deuterated succinate or acetate as the carbon source because these are less expensive. Despite the widespread use of BL21, we found no data on the effect of different minimal media and carbon sources on BL21 growth. In this study, we assessed the growth behavior of E. coli BL21 in minimal media with different gluconeogenic carbon sources. Though BL21 grew reasonably well on glycerol and pyruvate, it had a prolonged lag-phase on succinate (20 h), acetate (10 h), and fumarate (20 h), attributed to the physiological adaptation of E. coli cells. Wild-type strain NCM3722 (K12) grew well on all the substrates. We also examined the growth of E. coli BL21 in minimal media that differed in their salt composition but not in their source of carbon. The commonly used M9 medium did not support the optimum growth of E. coli BL21 in minimal medium. The addition of ferrous sulphate to M9 medium (otherwise lacking it) increased the growth rate of E. coli cultures and significantly increased their cell density in the stationary phase. [ABSTRACT FROM AUTHOR]

Aim: To study the neural mechanism by which electroacupuncture (EA) at RN12 (Zhongwan) and BL21 (Weishu) regulates gastric motility., Methods: One hundred and forty-four adult Sprague Dawley rats were studied in four separate experiments. Intragastric pressure was measured using custom-made rubber balloons, and extracellular neuron firing activity, which is sensitive to gastric distention in the dorsal vagal complex (DVC), was recorded by an electrophysiological technique. The expression levels of c-fos, motilin (MTL) and gastrin (GAS) in the paraventricular hypothalamic nucleus (PVN) were assayed by immunohistochemistry, and the expression levels of motilin receptor (MTL-R) and gastrin receptor (GAS-R) in both the PVN and the gastric antrum were assayed by western blotting., Results: EA at RN12 + BL21 (gastric Shu and Mu points), BL21 (gastric Back-Shu point), RN12 (gastric Front-Mu point), resulted in increased neuron-activating frequency in the DVC (2.08 ± 0.050, 1.17 ± 0.023, 1.55 ± 0.079 vs 0.75 ± 0.046, P < 0.001) compared with a model group. The expression of c-fos (36.24 ± 1.67, 29.41 ± 2.55, 31.79 ± 3.00 vs 5.73 ± 2.18, P < 0.001), MTL (22.48 ± 2.66, 20.76 ± 2.41, 19.17 ± 1.71 vs 11.68 ± 2.52, P < 0.001), GAS (24.99 ± 2.95, 21.69 ± 3.24, 23.03 ± 3.09 vs 12.53 ± 2.15, P < 0.001), MTL-R (1.39 ± 0.05, 1.22 ± 0.05, 1.17 ± 0.12 vs 0.84 ± 0.06, P < 0.001), and GAS-R (1.07 ± 0.07, 0.91 ± 0.06, 0.78 ± 0.05 vs 0.45 ± 0.04, P < 0.001) increased in the PVN after EA compared with the model group. The expression of MTL-R (1.46 ± 0.14, 1.26 ± 0.11, 0.99 ± 0.07 vs 0.65 ± 0.03, P < 0.001), and GAS-R (1.63 ± 0.11, 1.26 ± 0.16, 1.13 ± 0.02 vs 0.80 ± 0.11, P < 0.001) increased in the gastric antrum after EA compared with the model group. Damaging the PVN resulted in reduced intragastric pressure (13.67 ± 3.72 vs 4.27 ± 1.48, P < 0.001). These data demonstrate that the signals induced by EA stimulation of acupoints RN12 and BL21 are detectable in the DVC and the PVN, and increase the levels of gastrointestinal hormones and their receptors in the PVN and gastric antrum to regulate gastric motility., Conclusion: EA at RN12 and BL21 regulates gastric motility, which may be achieved through the PVN-DVC-vagus-gastric neural pathway.