Batch cultivation of B. megaterium for recombinant dextransucrase production
The B. megaterium strain MS941 transformed with the plasmid pMM1520dsrS which carries the gene of a dextransucrase from Leuconostoc mesenteroides  was chosen as the candidate strain for the investigation on heterologous dextransucrase production. The medium used was developed for an optimal growth of the B. megaterium strain investigated in this study . It contains per liter 33 g glucose monohydate, 5.0 g (NH4)2SO4, 2.2 g KH2PO4, 0.3 g MgSO4·7H2O, 0.5 g yeast extract, 2.0 mL trace element solution and 0.1 mL Sigma antifoam 204 per liter. The trace element solution contained 40 g MnCl2·4H2O, 53 g CaCl2·2H2O, 2.5 g FeSO4·7H2O, 2.5 g (NH4)6Mo7O24·4H2O and 2.5 g CoCl2· 6H2O.
At first, shaking flasks containing 50 mL of the medium were inoculated with a glycerol stock of the B. megaterium strain MS941 carrying the plasmid pMM1520dsrS (B. megaterium MS941dsrS) and cultivated overnight at 37°C and 250 rpm. Upon reaching an OD578 of approximately 3, 10 mL culture were added to 990 mL batch medium in a bioreactor (Biostat B2) and batch culture was carried out at 37°C, pH 5.2, 500 rpm and aeration of 1 L air/min. Analysis of biomass, glucose and metabolites were carried out as described in detail by Hollmann and Deckwer (2004). Biomass as cell dry weight (CDW) was calculated from measured OD value according to a linear relationship between OD and CDW that was determined in our previous works (data not published). Production of recombinant dextransucrase was induced by adding xylose at a concentration of 0.5% w/v after the OD578 reached about 0.3. For comparative 2-DE analysis, another batch culture without xylose induction was carried out under the same conditions.
For 2-DE analysis cell samples were taken from the xylose induced culture immediately before the induction (I0), then 1 h (I1), 6 h (I6) and 10 h (I10) after the induction. Similarly, after the OD578 reached about 0.3 the first cell sample (NI0) was taken from the non-induced culture. Further samples were then taken 1 h (NI1), 6 h (NI6) and 10 h (NI10) after the first sampling. Cells were immediately chilled in ice water after sampling, then centrifuged at 6500 rpm (Sorvall RT 6000B, DuPont) for 30 min at 4°C. Cell pellets were washed twice with phosphate-buffered saline (PBS) solution and stored at -80°C until use.
For the detection of the production and secretion of dextransucrase in B. megaterium, samples taken from the induced culture were centrifuged at 4°C and 5000 rpm for 10 min to obtain cell-free supernatants and cell pellets. The supernatants were centrifuged again at 4°C and 13000 rpm for 10 min to obtain protein sediments which contained the secreted dextransucrase as the secretion fractions. The cell pellets were resuspended with a lysis buffer (100 mM Na3PO4, 5 mg/mL lysozyme, pH 6,5 with H3PO4) and incubated at 37°C for 30 min and centrifuged at at 4°C and 13000 rpm for 10 min to separate the supernatant as the cell-associated soluble fractions from the sediments, which were then resuspended in 8M urea and centrifuged again to collect the supernatant as the cell-associated insoluble fractions. All three fractions were subjected to the determination of the activities of dextransucrase according to an activity staining method described by Malten et al . Briefly, proteins of the three fraction were separated with normal SDS-PAGE gels. DsrS which was still bound to the gels was renaturated and incubated with sucrose to catalyze the formation of dextran. The amount of formed dextran was set into relation to the catalytic activity of DsrS for the quantification of the activity.
Extraction and separation of intracellular protein of B. megaterium by 2-D IEF/SDS-PAGE
The 2-D IEF/SDS-PAGE gel electrophoresis (2-DE) method established in our laboratory for the proteomic analysis of different microorganisms  has been optimized for the separation of intracellular proteins of B. megaterium. To obtain raw protein extracts cell pellets were resuspended with a lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) dithiothreitol (DTT), 0.8% (w/v) Pharmalyte™ pH 3–10, and 5 mM Pefabloc, and disrupted by ultrasonication in a ice bath for 5 × 60 s and 2 × 30 s with a 30 s interval between each ultrasonic cycle for better cooling effect. Insoluble materials were separated by centrifugation at 13,000 g for 30 min at 4°C. For an improved 2-DE separation of B. megaterium proteins, raw protein extracts should be further treated to diminish other interfering cell-intrinsic components. Different purification methods such as membrane dialysis using the Mini Dialysis Kits, 1 kDa cut-off (Amersham Biosciences), or precipitation with various organic solvents have been tested. A phenol precipitation with subsequent acetone extraction resulted in the best 2-DE separation performance. Briefly, raw protein extracts were extracted with a TE-buffer (10 mM EDTA, pH 7.4) saturated phenol by vigorous shaking and incubation. Proteins form a white interphase between the phenolic and the aqueous phases. After discarding the aqueous phase and washing the protein interphase twice with Milli-Q water, proteins were precipitated with cold acetone (-20°C), washed additionally two times with cold acetone, air-dried and stored at -80°C until use.
Aliquots of protein pellets were diluted with an adequate volume of rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, 0.5% IPG buffer pH 4–7 and trace amount of bromphenol blue). The total protein concentration in the supernatant was determined by the Bradford method using the RotiQuant reagent (Bio-Rad) according to the manufacturer's instruction. Duplicate gels were analyzed for each sample and all the samples from the same batch culture were run under the exactly same conditions. The first-dimensional gel electrophoresis of isoelectric focusing (IEF) was run with the IPGphor Isoelectric Focusing System (Amersham Biosciences) at a temperature of 20°C. 250 μg of each protein sample were loaded onto Immobiline DryStrip gels (IPG strips) of pH 4–7 by in-gel rehydration. IEF was performed with the following parameters: 30 V × 6 h, 60 V × 6 h, 200 V × 1 h, 500 V × 1 h, 1000 V × 1 h, gradient from 1000 V to 8000 V within 30 min, then 8000 V × 10 h. The second-dimensional gel electrophoresis of SDS-PAGE was carried out using the vertical slab separation unit Ettan Dalt II System and pre-cast polyacrylamide gels Ettan Dalt II Gel 12.5% (Amersham Biosciences). Prior to SDS-PAGE the IPG strips were equilibrated in the SDS equilibration buffer as recommended in the user's manual provided by the manufacturer for the pre-cast gels. SDS-PAGE separation was performed at 25°C in constant power mode as follows: 2 W/gel for 1 h and then 20 W/gel until the bromophenol blue dye front reached the bottom of the gel. Subsequently, gels were stained using Brilliant Blue G-Colloidal Concentrate (Sigma, Saint Louis, Missouri, USA) by fixing in a glacial acetic acid : methanol : water (7:40:53) solution for 1 h; staining overnight with Brilliant Blue G-Colloidal according to the manufacturer's instruction and rinsing several times with Milli-Q water. The gels were then scanned with a UMAX PowerLook III scanner at 300 dpi resolution to acquire the gel images. Computer analysis of the gels for protein spot detection, matching and quantification were performed with the Phoretix 2D Advanced Software Version 2003.02 (Phoretix, Newcastle upon Tyne, UK). Exactly same parameters such as sensitivity and operator size were used for spot detection. The same background subtraction method "lowest on boundary" was applied for all gels. User seeds were added to help spot matching between gels. To overcome the problem of electrophoretic variations between gels, minor manual adjustments during spot detection and matching were performed when it was obviously necessary. Average values of protein spot volume intensities and their corresponding standard deviations were calculated to characterize expression changes of proteins. Protein spot intensity was defined as the normalized spot volume which is the ratio of the single spot volume to the total spots volumes on a 2-D gel. Normally only proteins showing more than 2-fold increase or decrease in expressions were considered to be up- or down-regulated.
Protein digestion and mass spectrometric analysis
Protein spots cut out from 2-DE gels were subjected to in-gel digestion with trypsin according to a method described previously  with some modification, namely, after rinse with water and dehydration with acetonitrile gel pieces were directly tryptic digested without further treatment. Peptides obtained were then extracted and purified with reversed-phased C18 ZipTips (Millipore, Bedford, USA).
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a Bruker Ultraflex time-of-flight mass spectrometer (Bruker Daltonics GmbH, Germany) and nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) with a Q-TOF 2 mass spectrometer (Micromass, Manchester, England) equipped with a nanospray ion source were carried out as described by Wang et al .
Protein identification by homologue protein searching
Peptide masses obtained from MALDI-TOF MS analysis were used for cross-species homologue protein searching in the public protein databases NCBInr and SWISS-PROT/TrEMBL by peptide mass fingerprinting. The search program Mascot (Matrix Science Ltd., UK, http://www.matrixscience.com) was used and search parameters were given as follows: trypsin was the digestion enzyme used, one missed cleavage sites was allowed, cysteine was modified by iodoacetamide and methionine was assumed to be partially oxidized. All peptide mass values are monoisotopic and the mass tolerance was set at 100 ppm.
MS/MS spectra of selected peptide precursors from ESI-QqTOF MS/MS analysis were enhanced using the Max Ent 3 software (Micromass), followed by automatic or manual sequencing using the PepSeq program of the software package Masslynx™ Version 3.5 (Micromass). Peptide sequences obtained were merged and submitted for similarity searching of homologous proteins using the protein database search program MS BLAST http://dove.embl-heidelberg.de/Blast2/msblast.html[7, 16] with the scoring matrix PAM30MS and against the comprehensive non-redundant protein sequence database nrdb95. No constraints on protein molecular weights (Mw), isoelectric point (pI) or species of origin were imposed.