Post-induction Temperature Research Articles

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

A statistical approach for optimization of RANKL overexpression in Escherichia coli: Purification and characterization of the protein

Receptor activator of nuclear factor-κB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3mM, a post-induction temperature of 25°C and a post-induction time of 6.5h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-α, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1±0.5μM.

Optimizing dimodular nonribosomal peptide synthetases and natural dipeptides in an Escherichia coli heterologous host

Nonribosomal peptides are an important class of natural products that have a broad range of biological activities. Their structural complexity often prevents simple chemical synthesis, and production from the natural producer is often low, which deters pharmaceutical development. Expression of biosynthetic machinery in heterologous host organisms like Escherichia coli is one way to access these structures, and subsequent optimization of these systems is critical for future development. We utilized the aureusimine biosynthetic gene cluster as a model system to identify the optimal conditions to produce nonribosomal peptides in the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible T7 promoter system of pET28. Single reaction monitoring of nonribosomal products was used to find the optimal concentration of IPTG, postinduction temperature, and the effect of amino acid precursor supplementation. In addition, principle component analysis of these extracts identified 3 previously undiscovered pyrazine products of the aureusimine biosynthetic locus, highlighting the utility of heterologously expressing nonribosomal peptide synthetases to find new products.

Over expression of the Interferon β-1b by optimizing induction conditions using response surface meth-odology

High expression of therapeutic proteins is crucial for industrial scale manufacturing. In the present study, induction conditions for the production of interferon β-1b by recombinant Escherichia coli BL21 (DE3) were optimized using response surface methodology based on the central composite design (CCD). The induction conditions included isopropyl β -Dthiogalactopyranoside (IPTG) concentration, induction start time (OD600 nm), post-induction time and temperature. The optimal set of culture conditions for high interferon β-1b was determined: 0.7 mM IPTG, an induction start time (OD600 nm) of 0.58, a post-induction time of 5h, and a post-induction temperature of 37°C. A high level of interferon beta-1b protein was expressed in the optimized conditions, which accounted for about 40.2 % of the total cell proteins.

Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies

Tumor necrosis factor-α (TNF-α) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron’s disease, stroke, and atherosclerosis. Thus, inhibition of TNF-α is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-α inhibitors. In this work, induction conditions for the production of human TNF-α fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD600nm), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-α. An 11% increase of TNF-α production was achieved after determination of the optimum induction conditions: OD600nm prior induction 0.55, a post induction temperature of 25°C, an IPTG concentration of 1mM and a post-induction time of 4h. We have also studied TNF-α oligomerization, the major property of this protein, and a Kd value of 0.26nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable Kd value for protein trimerization probably due to the complexibility of our model.

The efficacy of prewarming on post-induction core temperature and thermoregulatory response under general anesthesia

The efficacy of prewarming on post-induction core temperature and thermoregulatory response under general anesthesia

Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology

Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in shake flask culture.

Evolutionary Remodeling of βγ-Crystallins for Domain Stability at Cost of Ca2+ Binding

The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across βγ-crystallin members critical for their specialized functions.

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24 ◦ C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

Anti-lipopolysaccharide factors (ALFs) are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Escherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, (NH4)2SO4 and KH2PO4, were further optimized via the central composite design (CCD). Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside (IPTG) concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L (NH4)2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% (without optimization) to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 10.5% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.

An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.

GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli

Human phospholipid scramblase 1(hPLSCR1), when expressed in E. coli (BL-21 DE3), forms inclusion bodies that are functionally inactive. We studied the effects of various stress inducing agents and chaperones on soluble expression of hPLSCR1 in E. coli (BL-21 DE3). Addition of 3% (v/v) ethanol before induction and decreasing the post-induction temperature to 15 degrees C increased the solubility of hPLSCR1 to approximately 10 and approximately 15% respectively. Presence of groES-groEL complex solubilized the hPLSCR1 to approximately 30% of the total hPLSCR1. Absence of groES-groEL did not improve the solubility of hPLSCR1 suggesting that groES and groEL are the essential chaperones for the correct folding of hPLSCR1 when over-expressed in E. coli.

Effect of prewarming on post-induction core temperature and the incidence of inadvertent perioperative hypothermia in patients undergoing general anaesthesia

BackgroundInadvertent perioperative hypothermia (IPH) occurs in many patients because warming techniques are insufficient to counteract thermal redistribution resulting from the ablation of thermoregulatory vasoconstriction associated with anaesthesia. We tested the efficiency of a preoperative forced-air warming (FAW) device (Bair Paws®) in preventing IPH. MethodsSixty-eight adult patients undergoing spinal surgery under general anaesthesia were randomized to receive either normal care or prewarming for 60 min, at 38°C, using the Bair Paws® system. All patients received routine FAW intraoperatively. ResultsThirty-one patients were prewarmed and 37 patients were in the control group. There was a 0.3°C smaller decrease in mean core temperature in the prewarmed group at 40, 60, and 80 min post-induction (P≤0.05). Temperature was maintained above the hypothermic threshold of 36°C in 21 (68%) patients in the prewarmed group, compared with 16 (43%) patients in the control group (P<0.05). ConclusionsPreoperative warming using the Bair Paws® system results in smaller decreases in core temperature intraoperatively and less IPH in patients undergoing spinal surgery under general anaesthesia.

Optimization of culture conditions to enhance cis-epoxysuccinate hydrolase production in Escherichia coli by response surface methodology

The effect of culture conditions on the cis-epoxysuccinate hydrolase (CESH) production in recombinant Escherichia coli BL21 was investigated using response surface methodology (RSM), which was based on rotatable central composite design. The optimization of seed conditions consisted of a total of 13 experiments involving 4 star points and 5 replicates at the central points, while the optimization of induction conditions consisted of a total of 31 experiments involving 8 star points and 7 replicates at the central points. The optimum predicted culture conditions for maximum expression of recombinant CESH were found to be comprised of 11.75 h seed age, 4.13% (v/v) inoculation level, OD 600 0.2 induction-starting time, 2.53% (w/v) lactose, 24.29 °C post-induction temperature and 27.56 h post-induction time, with a predicted CESH activity of 40,460 U/g, which was very close to the experimental CESH activity of 40,129 U/g resulting in 4.6-fold increment after optimization.

Periplasmic expression and recovery of human interferon gamma in Escherichia coli

A synthetic human interferon gamma (hIFN-γ) gene was fused to SP1 and SP3, two Sec-dependent artificial signal peptides to transport the hIFN-γ to the periplasm of Escherichia coli BL21-SI. The processing efficiency of both SP1-hIFN-γ and SP3-hIFN-γ was dependent on the culture medium as well as the post-induction temperature. Both precursors were processed completely when cells were cultured using minimal medium and a post-induction temperature of 32.5°C, and only the processed hIFN-γ was detected. The SP3 signal peptide was more efficient than SP1 for the secretion of hIFN-γ. Sixty percent of the total hIFN-γ was secreted to the periplasm using the SP3 signal peptide and a post-induction temperature of 20°C. Using Tris–sucrose–dithiothreitol (TSD) hypertonic buffer, the periplasmic soluble hINF-γ was recovered with a purity of 85%.

The Cyclodextrin Glycosyltransferase of Paenibacillus pabuli US132 Strain: Molecular Characterization and Overproduction of the Recombinant Enzyme

The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the β-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37°C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19°C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed Vmax and Km values of 253 ± 36 μmol of β-cyclodextrin/mg/min and 0.36 ± 0.18 g/L, respectively.

A C-terminal Region of Signal Peptide Peptidase Defines a Functional Domain for Intramembrane Aspartic Protease Catalysis

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.

Soluble expression of a pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli

Microbial pro-transglutaminase (MTG) from Streptomyces mobaraensis was cloned and expressed for the first time as a soluble protein at high levels in Escherichia coli. According to SDS-PAGE, more than 90% of the transglutaminase was produced in soluble form. This high solubility was obtained either by using a lactose auto-induction medium at a cultivation temperature of 28 °C or a temperature shift strategy comprising of growth at 37 °C and a post-induction temperature of 24 °C in a conventional LB medium and IPTG as the inductor. Using the auto-induction procedure the specific activity of MTG was as high as 627 U g −1 CDM after activation by cleaving off the pro-sequence. Using shake flask cultivation, yields of 1460 U l −1 bioreactor volume were obtained. This amount of soluble MTG is sufficient to enable high-throughput screening of mutant libraries without the restraint of refolding. Preceding optimization experiments of the transglutaminase expression included variation of the inductor type, temperature after expression, the localization as well as the supply of rare t-RNAs. Almost exclusively insoluble inclusion bodies were formed by IPTG induction at 37 °C. Transport to the periplasm using a pelB leader sequence failed and the supply of rare t-RNAs by E. coli Rosetta(DE3) did not enhance the soluble fraction of the protein. Thirty percent soluble transglutaminase were obtained by lowering the growth temperature from 37 to 24 °C after induction with IPTG when a pelB leader sequence was used.

Excretory over-expression of Bacillus sp. G1 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: Optimization of the cultivation conditions by response surface methodology

Co-expression of cyclodextrin glucanotransferase (CGTase) from Bacillus sp. G1 ( cgt) with bacteriocin release protein (BRP) in Escherichia coli system resulted in the expression and excretion of the enzyme into the culture medium. The cultivation conditions were then optimized with the objective to enhance the production of extracellular recombinant CGTase using response surface methodology (RSM) that based on rotatable central composite design (CCD). The process consisted of a total of 50 experiments involving 10 star points and 8 replicates at the central points. The optimum predicted cultivation conditions for the maximum expression of extracellular recombinant CGTase were found to be comprised of: 20 °C post-induction temperature, induction-starting time when cell optical density is 0.3 at 600 nm, 1.0 mM xylose, 50 μM IPTG and 29 h post-induction time, with a predicted extracellular recombinant CGTase activity of 9144.28 U/ml. The experimental extracellular recombinant CGTase activity obtained was 9542.30 U/ml, which was very close to the predicted value. The expression of extracellular recombinant CGTase improved about 151-fold after the optimization was conducted.

Rapid Optimization of Antibotulinum Toxin Antibody Fragment Production by an Integral Approach Utilizing RC-SELDI Mass Spectrometry and Statistical Design

A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.