Vector library preparation
The RI encoding gene was designed for insertion into an expression vector library via site specific recombination reaction based on the Gateway® cloning technology by PCR end extension (with High Fidelity PCR mix from Fermentas) according Invitrogen's Gateway cloning manual (for details see: http://www.invitrogen.com). The PCR fragment was purified from 1% agarose with the QIAquick Gel Extraction Kit (Qiagen) and inserted into the pDONR201™ (Invitrogen) vector via Gateway® cloning "BP recombination". The whole Gateway® BP recombination reaction was transformed into the ccdB gene effect sensitive E. coli DH5α strain via calcium transformation. The halves of the transformation mixture were plated on Luria broth (LB) plates with 50 μg mL-1 of kanamycin. The pDONR201™ vector, containing the target gene, was purified with the GeneJET™ Plasmid Miniprep Kit (Fermentas) and used for "Gateway® LR" recombination for insertion of the target gene into to the destination protein expression library. The recombination mixture was transformed into the ccdB sensitive E. coli DH5α strain via calcium transformation and plated on LB solid medium with 30 μg mL-1 of chloramphenicol. The expression vectors, containing the target gene, were purified with the GeneJET™ Plasmid Miniprep Kit (Fermentas) and used for the following transformations.
Preparation of target protein expression strain library
The expression strain E. coli K12 RV308 (ATCC 31608) was first transformed with the reporter plasmid pibpfxsT7lucA previously described by Kraft et. al. , carrying a resistance for ampicillin, and plated on LB agar with ampicillin (100 μg mL-1). The expression strain E. coli RV308 pibpfxsT7lucA was co-transformed with the RI gene containing the cytoplasmic expression library; the transformants were plated on LB agar containing ampicillin (100 μg mL-1) and chloramphenicol (30 μg mL-1). Both transformations were based on the calcium temperature shock method. The cell stock was produced by washing the transformants from the surface of the agar plates with 2 mL of glucose-based mineral salt medium (MSM), containing the antibiotics and glycerol (25%). The collected cell suspensions having cell densities (OD600) of about 10 to 30 were aliquoted into sterile PCR stripes and stored at -70°C.
Transformations and plasmid propagations were performed on solid and liquid LB medium containing Bacto-Tryptone (10 g L-1), Bacto-yeast extract (5 g L-1), NaCl (10 g L-1), 15 g L-1 bacto agar (if solid medium) and the required antibiotics. Fed-batch and batch cultivations were performed in glucose-based mineral salt medium (MSM) with the following composition (per litre): Na2SO4 2 g, (NH4)2SO4 2.68 g, NH4Cl 0.5 g, KHPO4 14.6 g, NaH2PO4 × H2O 3.6 g, (NH4)2-H-citrate 1.0 g, and glucose 2.5 to 15 g. NaOH (40%) was used to adjust pH to 7.0 prior to the heat sterilisation. Semi-synthetic medium was based on the MSM with additional 10 g L-1 of yeast extract and 10 g L-1 casamino acids. Additionally, before cultivation on the mineral salt and semi-synthetic media the following sterile solutions were added: 2 mL L-1 of (1 M) MgSO4 and 2 mL L-1 of trace element solution with the following composition (per litre): CaCl2 × 2H2O 0.5 g, ZnSO4 × 7H2O 0.18 g, MnSO4 × H2O 0.1 g, Na2-EDTA 20.1 g, FeCl3 × 6H2O 16.7 g, CuSO4 × 5H2O 0.16 g, CoCl2 × 6H2O 0.18 g; as well as 100 μL L-1 of thiamine hydrochloride (1 M), 1 mL L-1 of ampicillin (100 mg mL-1) and 1 mL L-1 of chroamphenicol (30 mg mL-1). The feeding solution for fed-batch cultivations was based on fully formulated MSM with the required antibiotics and 550 g L-1 of glucose.
Fed-batch mode cultures and recombinant protein synthesis in 96 microwell plates
EnBase® technology (BioSilta Oy, Oulu, Finland) based fed-batch 96 microwell plate cultures were performed in 96 well flat bottom plates (Perkin Elmer). The wells of the plates were filled with heat sterilized gels, referred as - "bottom" and "top" phases . Firstly, 100 μL of "bottom" gels (1.5% of Bacto-agar, Difco) and 10% of potato starch (Sigma) were added. After solidification of the bottom gels, 50 μL of top-gels (3.25% Bacto-agar) were added. The wells for cultivation were filled with 150 μL of fully formulated sterile MSM containing 100 μg mL-1 of ampicillin and 30 μg mL-1 of chloramphenicol. For cultivation the plates were closed with a plastic lid. All microscale pre-induction cultures were performed by intensive shaking with a Variomag® Thermoshake (Inheco, Germany) at 37°C and 750 rpm (amplitude 1.5 mm). The inoculum plate with the gel-based EnBase® system was inoculated with 5 μL of glycerol stock cultures per well. Synchronization of all 45 RI expression strain cultures was performed in these plates in the batch mode in MSM containing 2.5 g L-1 of glucose at 37°C for 12 hours (overnight). After overnight cultivation at the obtained cell densities (OD600 about 4.5 ± 1.0) the release of glucose from starch was started by addition of 5 μL of amylase (BioSilta Oy, Oulu, Finland) to obtain a final concentration of 6 AGU L-1 (amyloglucosidase units per liter). 1 AGU is the amount of enzyme which releases 1 μmol min-1 of maltose).
Additionally the culture suspensions were supplemented with 5 μL of NH4OH (25%). The inoculum cultures were cultivated for another 12 hours under glucose limitation at 37°C. After the initial cultivation 5 μL of synchronized and adapted precultures were transferred to the gel-containing wells of a new microwell plate (with 6 AGU L-1), from the beginning possessing linear glucose auto-release and cultivated for 9 hours at 37°C. Target gene expression was induced after 9 hours of cultivation at OD600 = 12 ± 0.5 by addition of 5 μL of IPTG, dissolved in fully formulated medium, to achieve a final concentration of 0.5 mM. At the time of induction also the cultivation temperature was shifted to either 37°C, 30°C, or 22°C, respectively. In all experiments the cultures were harvested 7 h after induction.
Determination of cell growth in microwell plates
The initial (0 h time point) optical density (OD) of all microwell plate cultivation samples, was determined by measuring the turbidity with a Victor3
plate reader (PerkinElmer) with the following settings: wave length 490 nm, 15-20 sec shaking before plate reading. Cell densities from EnBase®
cultures were determined by 30 fold dilution of 5 μL broth samples in clear deionized water or cultivation medium in a final volume of 150 μL in clear, flat bottom 96 microwell plates (PerkinElmer). The OD490
obtained by using the Victor3
were recalculated to OD600
with a 1 cm path length using the following equation, obtained by a calibration curve:
where Df is the dilution factor.
Fed-batch mode cultivations in shake flasks
The EnBase® technology based fed-batch shake flask cultivations were performed in 1 L baffled Erlenmeyer flasks in 200 mL of MSM. These shake flasks contained 100 mL of the "Bottom gel" (10% potato starch, 5% Bacto-agar) and 75 mL of the "Top gel" (5% Bacto-agar). The inoculum for the production cultures was prepared by overnight batch cultivation at 37°C in 100 mL of MSM containing 7 g L-1 of glucose and the appropriate antibiotics. For inoculation the cultures were washed and resuspended in 50 mL of fully formulated sterile MSM without glucose. 10-12% from the total cultivation volume of culture suspension was transferred to MSM EnBase® cultivation flask to achieve final volume of 200 mL, with the appropriate antibiotics and no glucose. 1 mL of glucoamylase, diluted in sterile deionized water, was added to the cultivation medium, just after inoculation to obtain a final amylase concentration of 12 AGU L-1. The cultivations were performed at 37°C and 180 rpm on a Multitron shaker with an orbit of 2.5 cm (Infors). Product - 6 × His-MBP-RI synthesis was induced at three different time points (t1 to t3) which corresponded to the following specific growth rates: t1 = 4 h, OD600 = 2.0 ± 0.2, μ1 ≈ 0.33 h-1; t2 = 8 h, OD600 = 5.5 ± 0.2, μ2 ≈ 0.22 h-1; and t3 = 13 h, OD600 = 11 ± 0.2, μ3 ≈ 0.1 h-1. Induction was performed by addition of IPTG (1 M) to achieve a final concentration of 0.5 mM. The protein was produced for 4 h, at 37°C, shaking at 180 rpm.
Batch mode cultivations in the shake flasks
The inoculums for batch protein production in the shake flasks were prepared by overnight batch cultivation of the selected clone in 100 mL volume shake flask with 10 ml of LB at 37°C. For protein production, 1% of the corresponding inoculum culture was transferred either to fresh LB, or to mineral salt medium, or to a semi synthetic medium, both containing 10 g L-1 of glucose, to a final volume of 200 mL in 1 L baffled Erlenmeyer shake flasks. Cultures were cultivated at 37°C and 180 rpm until they reached the induction point, corresponding to a cell density of OD600 = 1 ± 0.05. Induction was performed by addition of IPTG (1 M) to a final concentration of 0.5 mM. The MBP-RI fusion was synthesized for 4 h, at 37°C.
Batch and Fed-batch cultivations were performed in a 10 L working volume Biostat C bioreactor (with MFCS/win 2.0 supervisory system, B. Braun Biotech, Melsungen, Germany) with the following parameters: the pO2 was maintained at 30% by adapting the stirrer rate and automatic regulation of the air flow (from 0 to 30 liters per min), the cultivation temperature was 37°C (before and after induction), pH was controlled at 7.0 ± 0.1 by addition of NH4OH (25%) or H3PO4 (2 M). MSM containing 15 g L-1 of glucose was used in the batch production process.
The fed-batch cultivations were started with a volume of 8.5 L of MSM, and contained 4.5 g L-1 and 15 g L-1 of glucose, respectively. The feeding was controlled by the Biostat software (version 4.62).
Exponential feeding profiles were programmed to maintain a specific growth rate of μ ≈ 0.22 h-1
. The feeding profiles were calculated with the following equations:
is the initial feeding rate [L h-1
is the specific growth rate [h-1
] to be maintained during feed operation, and t
is the time after feed start [h]. The initial feeding rate was calculated from the mass balance on substrate according to.
are the cell dry weight (CDW) [g L-1] and the culture volume [L] at the time of the feeding start, respectively, S
[g L-1] is the substrate concentration in the feeding solution, and Yx/s is the yield coefficient (g CDW per g of glucose). Y
in all calculations was 0.3 g g-1 as calculated from batch fermentations.
Before initiation of the fed-batch mode cells were cultivated as a batch until OD600 ≈ 6 or ≈ 22, respectively. The biosynthesis of the product was induced during the fed-batch mode at OD600 ≈ 9 or OD600 ≈ 31, respectively. The specific growth rate at the time of induction was the same in both cases, 0.22 h-1. After induction by 1 M IPTG cultivations were continued for 4 h at 37°C under continuation of the exponential feed function. In the batch process, performed as a control, 6 × His-MBP-RI target protein synthesis was induced at OD600 ≈ 5 (μ ≈ 0.45 h-1) and the culture was continued for 4 hours at 37°C.
Luciferase assay-target protein misfolding stress monitoring
determination the analyzed bacterial cultures were separated from the cultivation medium by centrifugation in a microcentifuge (14000 rpm, 5 min). The cell pellet was resuspended in 0.9% NaCl and diluted to achieve 8 × 107
(corresponding to 0.10 OD600
) in a final suspension volume of 200 μL. The whole 200 μL suspensions were transferred to the wells of a 96 microwell plate (Greiner) with transparent bottom. Then 100 μL of fresh reaction buffer (25 mM tricine, 15 mM MgCl2
, 5 mM ATP, 7 mM beta-mercaptoethanol, 0.5 mg mL-1
bovine serum albumin, 13 mM D-luciferin Na-salt, pH 7.8) were added and the luminescence was measured with a Victor3
multilable counter (Wallac) every 10 min at 25°C over a total time of 60 min. Blank samples were included (expression strain cultures cells cultivated without induction). The "true" luminescence values were calculated for each sample from the average of the measured values during the plateau phase by applying the formula:
where Tv - True, Mv - Measured and Bv - Blank luminescence values calculated by formula Bv = (Blank signal) × Df × N, Df - dilution factor and N - normalization factor for the cells amount corresponding to cell density of 1 at OD600.
For visualization and quantification of the soluble and insoluble protein fractions from microwell plate cultures, normalised amounts of cellular suspension were transferred to 1.5 mL Eppendorf tubes, harvested by centrifugation (10 min, 14000 rpm, 4°C) and the pellet was resuspended in 100 μL of lysis buffer (50 mM Tris-HCl pH 8.0, 0.1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 5 mM DTT, 0.1 mg mL-1 lysozyme). The whole cellular suspensions were sonicated for 10 sec with a Vibra cell™ sonicator (Sonic and Materials Inc., 2 mm diameter probe tip) at 4°C. The supernatant (soluble fraction) was collected after centrifugation (10 min, 14000 rpm, 4°C) and the pellet (insoluble protein fraction) was resuspended in 100 μL of lysis buffer without lysozyme. Samples for SDS-PAGE were prepared as follows: 25 μL of 5× SDS-PAGE loading buffer, 5 μL of 20 × DTT (Fermentas Ltd.) and 20 μL of deionized water were added to 50 μL of the respective protein suspensions in order to obtain a final sample volume of 100 μL. Samples were heated for 15 min at 95°C. 10 μL of sample was applied to each lane of a 10% SDS-PAGE gel.
Cell samples harvested from flask and bioreactor cultivations were resuspended in lysis buffer with the following ratio: 1 g of biomass with 5 mL of lysis buffer. Lysis was performed by sonication for 30 sec (Vibra cell™, Sonic and Materials Inc., 6 mm diameter probe tip) at 4°C. The lysate was distributed to 1 mL fractions and centrifuged (10 min, 14000 rpm, 4°C). The supernatant (soluble protein fraction) was collected and the insoluble protein fraction containing pellets from the 1 mL disrupted cell suspensions were resuspended as described above in 1 mL lysis buffer without lysozyme. 10 μL of soluble protein and cellular debris suspensions were taken for SDS-PAGE sample preparation. Final volumes of 100 μL of SDS-PAGE samples were obtained as described above and 10 μL of these suspensions were applied for the SDS-PAGE run.
Quantification of the target protein in soluble protein fractions was performed after separation on an Agilent 2100 bioanalyzer. Therefore the normalised crude extracts were 4-fold diluted in the buffer containing 50 mM of Tris-phosphate pH 8.0 and 1 mM EDTA. Evaluation of protein amounts on SDS-PAGE gels was performed by using TotalLab software (Total Lab Systems). The ribonuclease inhibitor activity in the normalised for the biomass quantity crude extract was determined by activity assay described by Blackburn et al. .