Scorpion stings around the world

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Bacterial strains

In our project, three derivatives of the Escherichia coli K12 strain, the Escherichia coli WK6 were used:
▪ Escherichia coli WK6 wild-type: WK6 wild-type Escherichia coli K12 strain {Δ(lac-pro), galE, strA, nal; F’ lacIq ZΔM15, pro+}.
▪ Escherichia coli WK6/pHEN6/NbF12-10: a clone harboring the recombinant plasmid pHEN6 with cDNA fragment encoding the fragment of the bispecific VHHF12-VHH10 of the NbF12-10 nanobody coming from a VH domain bank of dromedary lymphocytes [4].
▪ Escherichia coli WK6/pHEN6/CH10-12: a clone harboring the recombinant plasmid pHEN6 with cDNA fragment encoding the humanized form of the bispecific VHH10-VHH12 of the CH10-12 nanobody coming from a VH domain bank of dromedary lymphocytes [165].
The strains were obtained from the Therapeutic Molecules and Venom Laboratory of the Pasteur Institute of Tunisia (Laboratoire de Venins et Biomolécules Thérapeutiques, IPT). The recombinant strains produce the nanobody NbF12-10 and its chimeric format, the nanobody CH10-12. The wild-type strain, WK6, is used as a reference on the kinetic analysis of biomass production. For the NbF12-10 strain we used two clones, named NbF12-10 NN and NbF12-10 NO. The difference between the clones is the date of transformation, which were 24/02/2015 and 15/03/2016 for NO and NN, respectively. The second transformation was made due to the suspicion of the loss of the recombinant plasmid in the strain NbF12-10 NO.
The pHEN6 vector derived from the pBR322 (Figure II-1) uses the lac promoter to induce the production of the Nanobodies in the periplasmic space of the bacteria as the soluble format and with a hexa-Histidine (His6) tag. The pHEN6 vector also gives to the strain the resistance to a specific antibiotic (ampicillin).
Figure II-1. A schematic representation of the pHEN6 vector used in the Escherichia coli WK6 strains

Bacterial glycerol stock

A bacterial stock was prepared from the strains of the Pasteur Institute of Tunisia (IPT) for their storage at -80°C in single-dose cryovials containing glycerol as cryoprotectant. Two different methods were used, the first using the protocol of the hosting laboratory (Toulouse Biotechnology Institute, TBI), and the second following the instructions of the IPT.
The bacterial stock primarily used was the one prepared following the TBI protocol, the stock prepared with the IPT protocol was stocked as back-up.

TBI protocol

Under sterile conditions, a 100 mL baffled shake-flask containing 15 mL of LB broth and final concentration of 100 µg/mL of ampicillin was prepared and inoculated with a single colony picked up from the LB agar plates using a sterile tip and grown at 37°C and 150 rpm for 12 h in a shaking incubator.
A 500 mL baffled shake-flask containing 45 mL of LB broth and final concentration of 100 µg/mL of ampicillin was inoculated with 5 mL of the inoculum and grown at 37°C and 120 rpm for 12 h in a shaking incubator.
After the incubation, 20 mL of pure glycerol was added to the culture attaining 30% of final concentration of glycerol; 30 sterile Cryotube® vials of 1.8 mL of final volume were filled with the glycerol stock and stored at -80°C.
For each strain this procedure was followed, except for the Escherichia coli wild-type were ampicillin was not added to the culture.

IPT protocol

Under sterile conditions, a 100 mL baffled shake-flask containing 15 mL of LB broth and final concentration of 100 µg/mL of ampicillin was prepared and inoculated with a single colony picked up from the LB agar plates using a sterile tip and grown at 37°C and 150 rpm for 12 h in a shaking incubator.
A 1 L baffled shake-flask containing 99 mL of LB broth and final concentration of 100 µg/mL of ampicillin was inoculated with 1 mL of the inoculum and grown at 37°C and 120 rpm for 12 h in a shaking incubator.
After the incubation, 45 mL of the culture were mixed with 15 mL of pure glycerol, attaining 25% of final concentration of glycerol; 27 sterile Cryotube® vials of 1.8 mL of final volume were filled with the glycerol stock and stored at -80°C.
For each strain this procedure was followed, except for the Escherichia coli wild-type were ampicillin was not added to the culture.

Culture media

Inoculum

The inoculum was prepared in a solid phase first in LB agar, and then a liquid phase in LB broth. The compounds in Table II-1, with exception of the ampicillin, were dissolved in distilled water and autoclaved at 121°C for 20 min. Ampicillin was added to a final concentration of 100 µg/mL to the LB agar plates before pouring, and to the LB broth just before inoculation.
A cryovial was thawed and, using a sterile tip, streaks were made on a LB agar plate with one drop of the cryostock. The plates were incubated at 37°C for 12 h. A 100 mL baffled shake-flask containing 15 mL of LB broth was inoculated with a single colony of the LB agar plate and grown at 37°C and 120 rpm for 12 h in a shaking incubator.

Rich medium (Terrific Broth)

Terrific Broth (TB) was used as the Rich or complex medium (Table II-2). The TB was designed especially for the recombinant Escherichia coli strains, allowing for a high plasmid yield and extending the growth phase of the culture [166]. The tryptone and the yeast extract are the source of nutrients, the potassium phosphates act as a pH buffer and the glycerol is added as an additional carbon source.
The dry components were dissolved in 900 mL of distilled water. Glycerol was added and distilled water was added to a final volume of 1 L. The solution was autoclaved at 121°C for 20 min.

Defined medium (Minimal Medium)

The defined culture medium or Minimal Medium (MM) was developed especially for the culture of Escherichia coli strains by the TBI and up to 30 g/L of biomass can be obtained [167]. The MM must be done in several steps to avoid the precipitation of mineral ions.

Trace elements

Each trace element is prepared in a 1000-fold concentrated stock solution (Table II-3). The stock solution was sterilized with a 0.22 µm filter (Sartorius) into a 100 mL penicillin vial crimped and previously autoclaved.

Salts B

The salts B (Table II-4) were prepared in stock solution 1000-fold concentrated, except for the Magnesium sulfate (MgSO4∙7H2O) which was 500-fold concentrated [167]. The stock solution was sterilized with a 0.22 µm filter (Sartorius) into a crimped 100 mL penicillin vial previous autoclaved.
The pH of the Iron sulfate (FeSO4∙7H2O) solution was adjusted to 2.0 with HCl before filter sterilization.

Thiamine (Vitamin B1)

A stock solution of 10 g/L of thiamine (C12H17N4OS+, vitamin B1) was sterilized with a 0.22 µm filter (Sartorius) into a 100 mL penicillin vial crimped and previously autoclaved. The thiamine solution was stored protected from light at 4°C.

Salts A

The salts A (Table II-5) were dissolved in distilled water in approximately 75% of the final desired volume [167].
A solution of 100 g/L of citric acid was prepared in a volume equivalent to 3/50 of the final desired volume. The trace elements and the salts B were added at 1 mL per liter of final volume in the order shown in Table II-3 and Table II-4. Magnesium sulfate was added at 2 mL per liter.
The citric solution was added to the salts A and pH was adjusted at 6.8 with 28% ammonia. The solution was brought to the final desired volume and autoclaved 20 min at 121°C. Thiamine was added at 1 mL per liter before inoculation.

Glucose solutions

Glucose was used as a carbon source for the cultures in minimal medium (MM) in shake flask and on the batch mode in the bioreactor cultures. A solution of glucose at 300 g/L was prepared in distilled water and autoclaved at 121°C for 20 min. The concentration of the solution was determined after autoclaving by enzymatic analyzer and HPLC. The density of the solution was measured by a densitometer (DE40 density meter, Mettler Toledo) and used for the mass calculations [168].

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Ampicillin stock

Due to their antibiotic resistance, recombinant strain cultures were supplemented with ampicillin. A solution of 100 g/L of ampicillin was prepared with 70% ethanol and filter sterilized with a 0.22 µm filter under the laminar flux hood. The solution was stored in 1 mL aliquots at -20°C.

IPTG stock

Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used as the inducer of the lac operon for the expression of periplasmic proteins. A solution of 1 M of IPTG was prepared with distilled water and filter sterilized with a 0.22 µm filter (Sartorius) under the laminar flux hood. The solution was stored in 2 mL aliquots at -20°C.

Shake flask cultures (Batch mode)

The cultures in shake flask were carried out with either rich medium (TB) or defined medium (MM). Biomass production was monitored during cell growth and during protein expression. Protein production was checked at the end of the culture.
TB was supplemented with 1 g/L of glucose, and MM with 10 g/L of glucose. The cultures with the recombinant strains were supplemented with 100 µg/mL of ampicillin. All cultures were performed in 2 L baffled shake flasks containing 330 mL of culture medium.

Biomass production

The cultures in TB and MM were inoculated with 1 mL of inoculum and carried out at 37°C and 120 rpm in a shaking incubator (Infors HT). Optical density, biomass cell dry weight and residual glucose were monitored until stationary phase was attained.

Protein production

The cultures in TB and MM were inoculated with 1 mL of inoculum and kept at 37°C and 120 rpm in a shaking incubator (Infors HT). Protein expression was induced with 1 mM of IPTG when the cultures reached 0.6 AU in a 10 mm cuvette (or 0.12 AU in a 2 mm cuvette) in TB. Temperature was modified to 28°C in a step change and cultures were kept under agitation for 12 to 16 h.

Bioreactor cultures (Fed batch mode)

The cultures carried out in a fully instrumented bioreactor and followed three phases: cell growth in batch mode, cell growth in fed batch mode, and induction. Batch and fed-batch mode were conducted at 37°C, induction was carried out at a range of 28 to 37°C, depending on the experiment.
Batch mode was started with 1.5 L of MM at 7.5 to 12.5 g/L of glucose; the inoculation of the bioreactor was made with 100 to 200 mL of inoculum in MM at 5 g/L of glucose. At depletion of glucose, an exponential feed was started, imposing a specific growth rate of 0.38 h-1, which is about half the maximum specific growth rate of Escherichia coli WK6 when grown in rich medium.
The production of the recombinant protein started when IPTG was added to the culture during a second fed-batch phase. Protein expression was made with 1 mM of IPTG when biomass reached 21 to 30 g cdw/L (Figure II-2). At induction, temperature was modified (28 to 37°C) and the feed solution rate was set to 4.5 g/h of glucose, which imposed a specific growth rate inferior to 0.05 h-1.

Instrumentation

The experimental set-up of the cultures in bioreactor is represented in Figure II-3. The bioreactor used in the experiments is a Biostat B-DCU (Sartorius) in borosilicate glass and stainless steel, of 5 L of working volume. The bioreactor is fully instrumented with sensors of dissolved oxygen, pH, temperature, and pressure. The control and monitoring of these parameters are made by the BioPAT MFCS (Sartorius) acquisition system.
Temperature, pH and inlet gas are controlled by the acquisition system. Temperature was controlled to 37°C for the batch and fed-batch mode by the circulation of water in the external double-wall of the bioreactor. The pH probe (pH 405-DPAS-SC-K8S/325, pH 0-12, 0-130°C, Mettler Toledo) was calibrated with buffer solutions of 4.00 and 7.01 pH (Sigma-Aldrich) before sterilization of bioreactor.
During cultures, the pH was controlled to 6.8 by addition of a solution of 14% w/v ammonia by a peristaltic pump (102R, 1-10 rpm Watson Marlow). The pressure probe was used only to monitor pressure in case of overpressure. The inlet gas was connected to a sparger (maximum 30 NL/min), which injected the air at the bottom of the bioreactor. The outlet gas was passed through a condenser to avoid high rates of evaporation and sent to a gas analyzer.
The stirrer of the bioreactor has a range from 10 to 3000 rpm. The impellers were distributed as shown in Figure II-4. Two six-flat-blade Rushton turbines and a three-blade marine impeller were used in the configuration. During the batch phase the first two impellers are submerged and during fed-batch phase the third impeller is gradually used with the change of volume in the bioreactor.

Table of contents :

Introduction
I.1. SCORPION STINGS AS A HEALTH PROBLEM
I.1.1. Scorpion stings around the world
I.1.1.1. America
I.1.1.2. Africa
I.1.1.3. Asia
I.1.2. Scorpion venom
I.1.3. Global market of therapeutics and treatment cost
I.1.3.1. Serotherapy
I.1.3.2. Commercially available antivenoms
I.1.4. Production of antivenoms
I.2. BIBLIOGRAPHY RESEARCH METHODOLOGY
I.2.1. Scientific databases
I.2.2. Keywords
I.2.3. Searching profiles
I.2.4. Coupled searching profiles
I.3. QUANTITATIVE ANALYSIS OF THE WORKING DATABASE
I.4. QUALITATIVE ANALYSIS OF THE WORKING DATABASE
I.5. BIOPRODUCTION OF RECOMBINANT PROTEINS
I.5.1. Production of recombinant proteins in Escherichia coli
I.5.1.1. Protein secretion mechanism
I.5.1.1.1. Excretion to medium
I.5.1.1.2. Periplasmic expression
I.5.1.1.3. Inclusion bodies
I.5.1.2. Protein inducer
I.5.1.3. Selection marker
I.5.1.4. Purification
I.5.1.5. Culture media
I.5.1.6. Culture conditions
I.5.1.7. Large-scale production
I.5.1.7.1. Cultivation processes
I.5.1.7.2. Downstream process
I.5.1.8. Emerging pharmaceutical applications of recombinant proteins
I.5.2. Bioproduction of scorpion toxins and antivenoms
I.5.2.1. Scorpion toxins
I.5.2.2. Scorpion antivenom
I.6. OBJECTIVE OF THIS PHD PROJECT
II.1. BACTERIAL STRAINS
II.2. BACTERIAL GLYCEROL STOCK
ii Susana María Alonso Villela
II.2.1. TBI protocol
II.2.2. IPT protocol
II.3. CULTURE MEDIA
II.3.1. Inoculum
II.3.2. Rich medium (Terrific Broth)
II.3.3. Defined medium (Minimal Medium)
II.3.3.1. Trace elements
II.3.3.2. Salts B
II.3.3.3. Thiamine (Vitamin B1)
II.3.3.4. Salts A
II.3.4. Glucose solutions
II.3.5. Ampicillin stock
II.3.6. IPTG stock
II.4. SHAKE FLASK CULTURES (BATCH MODE)
II.4.1. Biomass production
II.4.2. Protein production
II.5. BIOREACTOR CULTURES (FED BATCH MODE)
II.5.1. Instrumentation
II.5.2. Gas analyzer
II.5.3. Bioreactor feed solution
II.6. PHYSICAL AND BIOCHEMICAL ANALYSES
II.6.1. Optical density
II.6.2. Biomass cell dry weight
II.6.3. Microscopic analyses
II.6.4. Residual glucose
II.6.5. HPLC
II.7. NANOBODY EXTRACTION, PURIFICATION AND QUANTIFICATION
II.7.1. Osmotic shock
II.7.2. IMAC
II.7.3. Protein standards
II.7.4. Gel electrophoresis
II.7.5. Nanodrop (Absorbance)
II.7.6. Bradford Assay (Absorbance)
II.7.7. Image J (Densitometry)
II.8. DATA TREATMENT
II.8.1. Mass and energy balances
II.8.1.1. Volume
II.8.1.2. Gas analysis
II.8.1.2.1. Balance for Nitrogen
II.8.1.2.2. Balance for Oxygen
II.8.1.2.3. Balance for Carbon dioxide
II.8.1.2.4. Respiratory quotient
II.8.2. Carbon balance
II.8.3. Redox balance
II.8.4. Data smoothing
II.8.5. Enhancement factor
II.8.6. Microscopic observations
II.9. KINETIC MODEL OF THE NANOBODY PRODUCTION
III.1. STRAIN SCREENING
III.1.1. Culture study approach
III.1.2. Biomass characterization
III.1.2.1. Correlation coefficient between different biomass measurement methods
III.1.2.2. Evolution of the metabolites
III.1.2.3. Specific growth rate
III.1.2.4. Biomass yield
III.1.2.5. Acetic acid
III.1.3. Protein expression
III.1.4. Morphological analysis
III.1.5. Conclusion
III.2. BIOREACTOR CULTURES
III.2.1. Culture strategy
III.2.2. Microbial culture
III.2.2.1. Presentation of cultures
III.2.2.1.1. Reference cultures BR01 and BR09: Escherichia coli WK6
III.2.2.1.2. BR02 to BR05: Escherichia coli CH10-12 – short induction times (1st series)
III.2.2.1.3. BR06 and BR07: Escherichia coli CH10-12 – longer induction times (2nd series)
III.2.2.1.4. BR08: Escherichia coli NbF12-10 NN
III.2.2.2. Overall performance of bioreactor cultures
III.2.2.3. Oxygen transfer
III.2.2.3.1. Abiotic conditions
III.2.2.3.2. Biological conditions
III.2.2.3.3. Biological enhancement coefficient, E
III.2.3. Biotic variables
III.2.3.1. Mass and Energy balances
III.2.3.2. Biomass
III.2.3.2.1. Specific growth rate
III.2.3.2.2. Biomass yield
III.2.3.2.3. Morphological analysis
III.2.3.3. Acetic acid
III.2.3.4. Citric acid
III.2.3.5. Gas analysis
III.2.3.6. Respiratory coefficient
III.2.4. Nanobody
III.2.4.1. Short induction duration cultures (1st series)
III.2.4.2. Long induction duration cultures (2nd series)
III.2.4.3. Secondary proteins
III.2.4.4. Discussion
III.2.5. Conclusion
III.3. KINETIC MODEL
III.3.1. Kinetic models for recombinant proteins
III.3.2. Temperature effect in specific productivity of the nanobody production
III.3.3. Conclusion
Conclusions and perspectives
References

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