Auto-induction Medium Research Articles

Expression of soluble moloney murine leukemia virus-reverse transcriptase in Escherichia coli BL21 star (DE3) using autoinduction system.

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.

Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis

N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR.Key points• A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified.• The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum.• Synthesis of acyl amino acids was achieved in good yields.

Overproduction and purification of Mycobacterium tuberculosis WhiB3 in Escherichia coli is enhanced by co-expression with trigger factor chaperone

Members of the WhiB-like (Wbl) family of proteins are found in Acintomycetes and are somewhat recalcitrant to overproduction as soluble proteins in the laboratory protein expression workhorse Esherichia coli. The aim of this study was to evaluate the effects of culture conditions and co-expression of the chaperone protein, trigger factor (TF), on the soluble production of recombinant Mycobacterium tuberculosis (Mtb) WhiB3. A pET28a derived expression plasmid coding for His6-WhiB3 was created and the effects of varying the concentration of inducer (IPTG), the timing of induction, the nature of the inducer (auto-induction medium) and the temperature of the cultivation on the production of soluble His6-WhiB3 were tested. Whilst His6-WhiB3 protein was readily detected, the overwhelming majority of the protein was present in the insoluble fraction of cell-free extracts. However, co-expression of the tig from pTf16, coding for TF, increased His6-WhiB3 solubility dramatically, facilitating its isolation by affinity chromatography. Purified His6-WhiB3 was shown to be monomeric, and UV–visible spectra suggested that ∼10% of the isolated protein possessed a [4Fe–4S] cluster. The secondary structural properties of His6-WhiB3 were altered by acquisition of an iron-sulfur cluster. By developing a protocol to readily overproduce and purify WhiB3, this study paves the way for future structure-function experiments.

Heterologous expression of CYP81A6 from rice (Oryza sativa) in Escherichia coli and structural analyses of bensulfuron‐methyl metabolites

AbstractHerbicides are often selective and kill only weeds without affecting crops. In rice, CYP81A6 is responsible for tolerance toward acetolactate synthase herbicides such as bensulfuron‐methyl (BSM). CYP81A6 is thought to metabolize BSM into a nontoxic metabolite, that is, O‐demethylated BSM. However, direct evidences for this reaction have not been reported, likely due to difficulties in the heterologous expression of plant P450s in bacterial expression systems. In this study, we established CYP81A6 expression in Escherichia coli via modification of its N‐terminal basic region coding sequence, optimization of cultivation temperature, and selection of E. coli strains. Heterologous production of CYP81A6 was evaluated by the conversion of BSM in autoinduction medium after the culture, compared to that with an empty vector control. The optimized heterologous production condition of rice CYP81A6 in E. coli using 48‐well plates involved replacement of the N‐terminal basic region with that of CYP2C3, the use of E. coli BL21(DE3) as an expression host, and culture at 34°C. Further, co‐expression of gene encoding glucose dehydrogenase enhanced the conversion of 0.5 mM BSM from 58.7 to 73.1% in the optimized condition. In the optimization process, we detected two metabolites, M1 and M2. Mass spectrometry and 1H nuclear magnetic resonance analyses indicated that M1 was O‐demethylated BSM. Although we could not isolate M2 because of its low abundance in the medium, mass spectrometry analysis suggested that it was the monooxygenated (+O) form of BSM. Overall, these results confirm that CYP81A6 catalyzes the formation of O‐demethylated BSM from BSM.

3,3\u2032-Thiodipropionic acid (TDP), a possible precursor for the synthesis of polythioesters: identification of TDP transport proteins in Variovorax paradoxus TBEA6

3,3′-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

Whole Cell Biotransformation of 1-dodecanol by Escherichia coli by Soluble Expression of ADH Enzyme from Yarrowia lipolytica

In this study, alcohol dehydrogenases (ADH) enzymes from Yarrowia lipolytica were investigated for the cloning, soluble expression, and biotransformation of 1-dodecanol to 1-dodecanal, which reaction was thermodynamically unfavorable. Sole expression of ADHs in Escherichia coli did not produce soluble form of cytosolic protein, in spite of the effort to solubilize ADH protein by optimizing IPTG concentration, temperature, and auto-induction medium. Eventually, the active form of soluble ADH proteins was successfully obtained through the co-expression of ADH with chaperone protein in pG-KJE8 vector. After analyzing the individual sets of optimization, it was determined that pET-28a(+)::adh coexpression with the pG-KJE8 molecular chaperone in LB medium with 0.1 mM IPTG, 4 mg/mL arabinose, and 2 ng/mL tetracycline achieved optimum expressions against all of the five ADH proteins. Finally, the whole cell biotransformation activity of ADH2 was determined in 1-dodecanol oxidation to 1-dodecanal, followed by further oxidation to 1-dodecanoic acid.

Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.

Combinatorial strategy towards the efficient expression of lipoxygenase in Escherichia coli at elevated temperatures

Lipoxygenases (LOXs) are a family of non-heme iron oxidoreductases, which catalyze the addition of oxygen into polyunsaturated fatty acids. They have applications in the food and medical industries. In most studies, the soluble expression of LOXs in microbes requires low temperature (< 20°C), which increases the cost and fermentation time. Achievement of soluble expression in elevated temperatures (> 30°C) would shorten the production phase, leading to cost-efficient industrial applications. In this study, a combinatorial strategy was used to enhance the expression of soluble LOXs, comprising plasmid stability systems plus optimized carbon source used for auto-induction expression. Plasmid stability analysis suggested that both active partition systems and plasmid-dependent systems were essential for plasmid stability. Among them, the parBCA in it resulted in the enzyme activity increasing by a factor of 2 (498 ± 13 units per gram dry cell weight (U/g-DCW) after 6-h induction). Furthermore, the optimized carbon source, composed of glucose, lactose, and glycerol, could be used as an auto-induction expression medium and effectively improve the total and soluble expression of LOX, which resulted in the soluble expression of LOX increased by 7 times. Finally, the soluble expression of LOX was 11 times higher with a combinatorial strategy that included both optimized plasmid partition and auto-induction medium. Our work provides a broad, generalizable, and combinatorial strategy for the efficient production of heterologous proteins at elevated temperatures in the E. coli system. KEY POINTS : • Soluble expression of lipoxygenase at 30°C or higher temperatures is industrially beneficial. • Strategies comprise plasmid partition and optimized auto-induction medium with glucose, lactose, and glycerol as carbon source. • Combinatorial strategy further improved LOX soluble expression at 30°C and 37°C.

Combinatorial ethanol treatment increases the overall productivity of recombinant hG-CSF in E. coli: a comparative study.

Human granulocyte colony-stimulating factor (hG-CSF) is a cytokine that regulates the proliferation, maturation, and differentiation of precursor cells to neutrophils. In the present study, we report the feasibility of inducing recombinant hG-CSF expression (rhG-CSF) in a pET vector system by combinatorial induction using low-concentration ethanol, IPTG, and lactose and auto-induction media (AIM). The coding sequence of hG-CSF transcript variant 2 was expressed in pET14 vector, and the effect of combinatorial induction was analyzed on inclusion body (IB) formation, biomass, protein purification, and bioactivity. Results showed that there was an inverse relationship between the temperature and soluble expression of rhG-CSF. Three-step washing with Triton-X, 2M, and 5M urea resulted in the maximum recovery of IBs. Combinatorial single-spike induction with IPTG, ethanol, and lactose in a batch culture led to a 3-fold increase in the expression of rhG-CSF. It was also observed that low concentration of ethanol (1-3% v/v) could be used in lieu of IPTG for inducing the rhG-CSF protein expression without adversely affecting biomass production. A 2.4-fold increase in productivity was obtained in LB-AIM media with combinatorial ethanol induction, and the overall yield of 2.8g/L rhG-CSF was found. The purified rhG-CSF was bioactive and increased the cellular proliferation of umbilical cord blood-derived mesenchymal stem cells (U-MSC) by 29%. In conclusion, our study shows that combined ethanol induction can enhance the expression of rhG-CSF with three-step washing for recovery of the proteins from IBs and a single-step purification of rhG-CSF by affinity chromatography. KEY POINTS: • Low concentration of ethanol (1-3%) could be used in lieu of IPTG for inducing rhG-CSF expression. • Combinatorial single-spike induction with IPTG, ethanol, and lactose improved rhG-CSF expression. • Purified rhG-CSF was bioactive and increased the proliferation of U-MSC.

Crystal structure and physicochemical characterization of a phytocystatin from Humulus lupulus: Insights into its domain-swapped dimer

Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

Small subunits can determine enzyme kinetics of tobacco Rubisco expressed in Escherichia coli.

Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) catalyses the first step in carbon fixation and is a strategic target for improving photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits, and its biogenesis requires multiple chaperones. Here, we optimized a system to produce tobacco Rubisco in Escherichia coli by coexpressing chaperones in autoinduction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics that are very similar to the carboxylation kinetics of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO2 affinity compared with Rubisco complexes that are assembled with other small subunits. Our E. coli expression system will enable the analysis of features of both subunits of Rubisco that affect its kinetic properties.

Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica

Photoautotrophic microalgae offer a great potential as novel hosts for efficient recombinant protein production. Nannochloropsis oceanica produces an extraordinarily high content of polyunsaturated fatty acids, and its robust growth characteristics, published genome sequence and efficient nuclear transformation make N. oceanica a promising candidate for biotechnological applications. To establish a robust and flexible system for recombinant protein production, we cloned six endogenous, potentially constitutive or inducible promoters from N. oceanica strain CCMP1779 and investigated their strength using monomeric Venus as reporter gene. Microscopic pre-screening of individual transformants revealed that the promoters of elongation factor (EF), tubulin (TUB) and nitrate reductase (NR) enabled high reporter gene expression. Comparative quantitative analyses of transformant populations by flow cytometry and qRT-PCR demonstrated the highest Venus expression from the EF promoter and the NR promoter if extended by an N-terminal 14-amino acid leader sequence. The kinetics of reporter gene expression were analysed during photobioreactor cultivation, achieving Venus yields of 0.3% (for EF) and 4.9% (for NR::LS) of total soluble protein. Since inducible expression systems enable the production of toxic proteins, we developed an auto-induction medium for the NR promoter transformants. By switching the N source from ammonium to nitrate in the presence of low ammonium concentrations, the starting point of Venus induction could be fine-tuned and shifted towards exponential growth phase while maintaining high recombinant protein yields. Taken together, we demonstrate that a model recombinant protein can be produced robustly and at very high levels in N. oceanica not only under constitutive but also under auto-inducible cultivation conditions.Key points• Nannochloropsis oceanica might serve as host for recombinant protein production.• Comparative promoter strength analyses were conducted for twelve different constructs.• Robust high-yield recombinant protein production was achieved under constitutive conditions.• The nitrate reductase promoter enabled protein production under auto-induction conditions.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BackgroundAuto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level.ObjectivesIn this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium.MethodsFirst, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression).ResultsAuto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture.ConclusionThe medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Expression and purification of soluble recombinant SapM from Mycobacterium tuberculosis

SapM from Mycobacterium tuberculosis is a secreted phosphatase critical for pathogen survival inside the host, representing an attractive target for the development of anti-tuberculosis drugs. The main limitation to biochemical and structural studies of SapM has been the lack of a suitable protocol to produce soluble recombinant protein. The aim of the present work was to produce SapM in Escherichia coli in a soluble and catalytically active form. We describe here the construct design, expression and purification of soluble SapM using Sarkosyl as a solubility-enhancing agent and auto-induction media. We demonstrate that solubilisation of the recombinant protein with Sarkosyl, and further purification, yields a catalytically active enzyme with high purity and monodisperse. The identity and molecular weight of the recombinant SapM was confirmed by mass spectrometry analyses, and we provide evidence that SapM behaves as a monomer in solution. Overall, this work lays the foundation for further studies to exploit SapM as a drug target, and provides a protocol for producing active and soluble recombinant enzymes that are hard to solubilise in E. coli.

Overexpression and characterization of TnCel12B, a hyperthermophilic GH12 endo-1,4-β-glucanase cloned from Thermotoga naphthophila RKU-10T

A putative cellulolytic gene (825 bp) from Thermotoga naphthophila RKU-10T was overexpressed as an active soluble endo-1,4-β-glucanase (TnCel12B), belongs to glycoside hydrolase family 12 (GH12), in a mesophilic expression host. Heterologous expression and engineered bacterial cell mass was improved through specific strategies (induction and cultivation). Hence, intracellular activity of TnCel12B was enhanced in ZYBM9 modified medium (pH 7.0) by 8.38 and 6.25 fold with lactose (200 mM) and IPTG (0.5 mM) induction, respectively; and 6.95 fold was increased in ZYP-5052 auto-inducing medium after 8 h incubation at 26 °C (200 rev min−1). Purified TnCel12B with a molecular weight of ~32 kDa, was optimally active at 90 °C and pH 6.0; and exhibited prodigious stability over a wide range of temperature (50–85 °C) and pH (5.0–9.0) for 8 h TnCel12B displayed great resistance towards different chemical modulators, though activity was improved by Mg2+, Zn2+, Pb2+ and Ca2+. Purified TnCel12B had affinity with various substrates but peak activity was observed toward barley β-glucan (1664 U mg−1) and carboxymethyl cellulose (736 U mg−1). The values of Km, Vmax, kcat, and kcatKm−1 were found to be 4.63 mg mL−1, 916 μmol mg−1min−1, 1326.7 s−1 and 286.54 mL mg−1 s−1, respectively using CMC substrate. All noteworthy features of TnCel12B make it an appropriate industrial candidate for bioethanol production and various other potential applications.

Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain

During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37°C to 15°C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass.

Expression of recombinant human granulocyte-colony stimulating factor in Escherichia coli using various induction methods

Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that has several therapeutic applications. It consists of 174 amino acids and manufactured by recombinant DNA technology. Until now, the Escherichia coli expression system is still become the first choice for producing recombinant proteins. It is because of this organism is simple to culture in low-cost medium and easy to scale up. In the course to find the most efficient way to produce a high yield of recombinant human G-CSF, we compared several types of medium with different induction methods. In this experiment, recombinant E. coli NiCo21(DE3) harbouring gene encoding rh-GCSF proteins were cultured in various media including auto-induction, non-induction, and IPTG-induction. To determine the protein expression profile, culture sampling was done every 12 h (up to 60 h). Then, the optical density at ʎ 600 nm was measured using UV spectrophotometer and rh-GCSF protein expression were characterized using SDS-PAGE and western blot analyses. ImageJ software was used to calculate the amount of rh-GCSF protein yield using Bovine Serum Albumin (BSA) with known concentration as a standard. Result of this experiment concluded that simple auto-induction medium from Imperial College could produce good amount of rh-GCSF proteins (117 µg/mL) with relatively low production cost and short incubation time.

High yield production of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed batch fermentations using a recombinant strain of Escherichia coli

The enzymatic degradation of rubber with the latex clearing protein (Lcp1VH2) from Gordonia polyisoprenivorans VH2, is a promising option as an environmentally friendly and economical solution to treat the enormous amount of rubber waste. Here we present a fed batch fermentation process on a 10 L scale, using E.coli C41 pET23a(+)::Hislcp1VH2 and a modified defined mineral salt medium, designed for high cell densities, for a proper synthesis of Lcp1VH2. Particularly, providing complex media components, as well as hemin, as precursor of the essential heme b cofactor, resulted in a 2.9-fold higher yield of active Lcp1VH2 with increased specific activity, due to a better occupancy of the enzyme with the cofactor. Based on this optimization, the fed batch fermentation with an initial glucose feed, followed by a lactose-glycerol feed, finally gained a cell dry weight of 60 g L−1 and a yield of 223 mg L−1 of soluble, active Lcp1VH2. Compared to a recently published fermentation process, which used a complex auto-induction medium, we significantly increased the biomass up to nearly 10-fold and the total Lcp1VH2 yield up to 3.7-fold. Thereby we reduced the costs for the medium by 75 %, taking the next step towards industrial production of rubber degrading enzymes.

In-silico design and production of a novel antigenic chimeric Shigella IpaB fused to C-terminal of Clostridium perfringens enterotoxin

The emergence of antibiotic-resistant phenotypes in Shigella serotypes and the high mortality rate, approximately one million dead annually, in affected patients announce a global demand for an effective serotype-independent vaccine against Shigella. This study aims to design, express, and purify a novel chimeric protein, as a serotype-independent vaccine candidate against Shigella containing full-length Shigella invasion plasmid antigen B (IpaB) and a C-terminal fragment (residues 194-319) of Clostridium perfringens enterotoxin (C-CPE) as a mucosal adjuvant. Several online databases and bioinformatics software were utilized to design the chimeric protein and the relative recombinant gene. The recombinant gene encoding IpaB-CPE194-319 was synthesized, cloned into pACYCDuet-1 expression vector, and transferred to E. coli Bl21 (DE3) cells. IpaB-CPE194-319 was then expressed in auto-induction medium, purified and characterized using MALDI-TOF-TOF mass spectrometry. Followed by subcutaneous injection of the purified IpaB-CPE194-319 to BALB/c mice, antigenicity of this chimeric protein was determined through performing dot-blot immunoassay on nitrocellulose membrane using mice sera. The outcomes of this study show the successful design, efficient expression, and purification of IpaB-CPE194-319 divalent chimeric protein under mentioned conditions. The obtained results also demonstrate the intrinsic antigenic property of IpaB-CPE194-319.

Expression, purification and stabilization of human serotonin transporter from E. coli

The serotonin transporter belongs to the family of sodium-chloride coupled neurotransmitter transporter and is related to depression in humans. It is therefore an important drug target to support treatment of depression. Recently, structures of human serotonin transporter in complex with inhibitor molecules have been published. However, the production of large protein amounts for crystallization experiments remains a bottleneck. Here we present the possibility to obtain purified serotonin transporter from E. coli. Fos-choline 12 solubilized target protein was obtained with a purity of >95% and a yield of 1.2 mg L−1 culture in autoinduction medium. CD spectroscopic analysis of protein stability allowed identifying CHS and POPX as stabilizing components, which increased hSERT thermostability by 7 °C. The kinetic dissociation constant KD of 2.8 μM (±0.05) for of the inhibitor Desipramine was determined with a ka of 10,848 M − 1 s-1 (±220) and a kd of 0.03 s–1 (±4.7 × 10-5).