Bacterial strains and culture conditions
All plasmids and bacterial strains used in this study are shown in Additional file 1: Table S1. E. coli Trans1-T1 (Transgen, Beijing, China) was used for cloning procedures. E. coli BL21 (DE3) (Novagen, Beijing, China) was used for expression of the recombinant proteins. Cells were incubated in Luria-Bertani (LB) broth (100 μg/mL ampicillin or 50 µg/mL kanamycin) at 37 °C.
Molecular biology techniques and plasmid construction
Restriction enzymes were obtained from Fermentas (Beijing, China) or New England Biolabs (Beijing, China). DNA polymerase LA Taq and T4 DNA ligase were obtained from Takara (Beijing, China). TIANpure Mini Plasmid and Gel purification kits were from Tiangen (Beijing, China). All enzymes and kit reagents were used according to the manufactures’ instructions. The primers used in this study are summarized in Additional file 1: Table S2.
Codon usage in X12345 for expression in E. coli (http://www.kazusa.or.jp/codon/) was optimized using the codon optimization tool JCat (http://www.jcat.de/) and the optimized codon gene was synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). The full-length codon-optimized PtXyl43 (denoted X12345 herein) was inserted into the XbaI and XhoI sites of pET-28(a) to generate the vector pETX12345.
We also designed nucleotide sequences encoding the blade deletion or circular X12345 mutants as shown in Fig. 2b. The mutant genes were amplified by conventional PCR techniques or by partially overlapping primer-based PCR (Tao and He 2004) and individually cloned into the NcoI/XhoI sites of a pET-28(a) vector to construct the plasmids as listed in Additional file 1: Table S1.
To prepare plasmids encoding the passenger-carrier constructs, DNA sequences containing in the following order from the 5′ to 3′ end: the NcoI cleavage site, X12345 without a stop codon, the nucleotide sequence encoding the flexible (GGGGS)3 linker, the nucleotide sequence encoding the TEV-protease cleavage site (ENLYFQG), and the EcoRI recognition site, were constructed and PCR amplified (with X12345 acting as the carrier in the final construct). After digestion with the corresponding restriction endonucleases (NEB or Fermentas, Beijing, China), segment was ligated into pET-28a(+) to construct the carrier plasmid pX12345LT. The GFP, AIO6, and chitinase genes from the plasmids pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA, USA), pET-28a-aiio-AIO6 (used as passenger plasmid in this study) (Zhang et al. 2011), and pCHIX (used as passenger plasmid in this study) (Xu et al. 2016), respectively, and the hIL-2 gene which synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China), were PCR amplified. All these resulting PCR products, none of which contained a stop codon, were introduced into EcoRI site immediately upstream of their 5′-terminus and NotI site immediately downstream of their 3′-terminus. After digestion with EcoRI and NotI (NEB or Fermentas, Beijing, China), these genes were each individually ligated into the carrier plasmid containing X12345 digested with the same restriction enzymes to construct the passenger-carrier plasmids pX12345–GFP, pX12345–CHIX, pX12345–AIO6, and pX12345–hIL2. GFP and hIL-2 genes also were ligated into pET28a(+) which was digested with the same enzymes to construct passenger plasmids pGFP and phIL2.
All plasmids were verified by sequencing, and then the confirmed plasmids separately were transformed into E. coli Trans1-T1 and BL21 (DE3) competent cell samples.
Protein expression
For expression of each recombinant protein, first, E. coli BL21 (DE3) samples each transformed with one of the expression plasmids individually were cultured in 50 mL LB broth with shaking at 200 rpm for ~ 14 h at 37 °C for use as a seed culture. Each culture was then diluted 1:100 with Terrific Broth (TB) medium, 50 μg/mL kanamycin, pH 6.4 and cultured with shaking at 200 rpm at 37 °C until the OD600 of the culture was 0.8. Then lactose was added [final concentration, 2% (w/v)] to induce expression of the target protein, and each culture was incubated for 0–48 h at 20 or 33 °C. Each cell pellet and culture supernatant were separated by centrifugation at 10,000×g for 10 min at 22 °C to avoid cold shock. Proteins in the supernatants were precipitated with two volume of ice-cold acetone, and then the acetone precipitation and cell pellets were subjected to SDS-PAGE (12% (w/v) acrylamide) and western blotting analysis. Culture growth was monitored by measuring optical density at 600 nm with the BioPhotometer plus of Eppendorf AG (Hamburg, Germany).
Subcellular fractionation
Osmotic shock was used to isolate the E. coli periplasm from the cytoplasm proteins after expression of the constructs (Neu and Heppel 1965). Briefly, recombinant cultures (0.8 mL) at various induction time points were taken and centrifuged to separate into medium (medium fraction, M) and cell pellets (cellular fraction, C). Cell pellets were resuspended thoroughly in 0.6 mL of hypertonic solution (30 mM Tris–HCl (pH 8.0), 20% sucrose and 1 mM EDTA) and shaked slowly at room temperature for 10 min. Following centrifugation at 10,000×g at 4 °C for 10 min, supernatants were saved (periplasmic fraction, P1), and pellet were resuspended in 0.6 mL of ice-cold 5 mM MgSO4 (hypotonic solution) and shaked slowly for 10 min on ice. After centrifugation at 10,000×g at 4 °C for 10 min, the shocked cells (spheroplasts faction, S) and the supernatants (periplasmic fraction, P2) were saved. To isolate the soluble and insoluble fractions, cell pellets (cell fraction, C) from 0.8 mL of culture were resuspended by 0.5 mL of BugBuster Protein Extraction Reagent and incubated on a shaking platform for 10–20 min at room temperature. After centrifugation at 4 °C at 13,000×g for 20 min, pellets (the insoluble fraction, IF) and supernatants (the soluble fraction, SF) were saved. All the supernatants, such as medium fraction, periplasmic fraction (P1 and P2) and soluble fraction, were mixed with equal volume of acetone, respectively, then placed on − 80 °C for 1 h followed by centrifugation at 13,000×g for 10 min, and the corresponding resulting pellets were saved. To ensure comparability between different fractions, the separated fractions pellets were resuspended in the same volume of PBS buffer as that was used to suspend the cell pellet (cellular fraction, C) with OD600 of 10. Then 5× SDS-PAGE sample loading buffer was added to each fraction, and the fractions were boiled at 100 °C for 10 min and centrifuged at 4 °C at 13,000×g for 10 min before loading on gels for SDS-PAGE and western blotting. The same volume of each supernatant was loaded per lane.
SDS-PAGE and western blotting
Proteins in the culture medium, a sucrose hypertonic solution, the periplasm were isolated by precipitation with two volumes of ice-cold acetone. Protein solution was mixed with equal volume of acetone and kept at − 70 °C for 30 min, − 20 °C for 1 h, and then centrifuged at 4 °C at 13,000×g for 15 min. The pellets were air dried for at least 1 h and then suspended in PBS buffer (7.4). After electrophoresis loading buffer was added, all samples were boiled at 100 °C for 5 min and then centrifuged at 13,000×g for 10 min, and 10 μL of each loaded onto a SDS-PAGE gel (TGX Stain-Free™ FastCast™ Acrylamide Kit, 12%, Bio-Rad, Cat. No. 161-0185). After electrophoresis, the proteins were transferred to Immobilon®-PSQ membrane (Millipore, 0.2 μm) for immunoblotting. To detect the His-tag, a mouse monoclonal anti-His antibody (Tiangen, Beijing, China) was used. GroEL was detected using a rabbit polyclonal antiserum against GroEL (Sigma, Shanghai, China). Monoclonal antibody against MBP was from Beijing Protein Innovation Co., Ltd (Beijing, China).
Mass spectrometry (MS)
The culture supernatant from E. coli BL21(DE3) that expressed X12345 for 48 h after lactose induction was loaded onto a pre-equilibrated Ni2+-NTA column and washed with 4 column volumes of elution buffer (pH 8.0) containing 20, 40, 60, 80, or 200 mM imidazole in 300 mM NaCl, 50 mM NaH2PO4 solution (flow rate: 1 mL/min). The protein fraction that was eluted with 60 mM imidazole was subjected to quadrupole time-of-flight (Q-TOF) MS at the Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Field emission scanning electron microscopy (FESEM)
After lactose induction for 8, 12 or 24, E. coli BL21(DE3)/pX12345 or BL21(DE3)/pET28a cells were harvested by centrifugation (2200×g, 5 min), and washed twice with PBS buffer (pH 7.0). Samples were fixed with 2.5% glutaraldehyde overnight at 4 °C, then washed three times with 0.1 M PBS (pH 7.2) for 10 min each, dehydrated through gradient ethanol elution [30, 50, 70, 85 and 95% (v/v)] for 15 min at each concentration, and followed by 100% (v/v) ethanol three times for 15 min each. Samples were critically point dried in a CO2 atmosphere (BAL-TEC CPD 030, Leica Microsystems GmbH, Wetzlar, Germany), sputter-coated with gold palladium by Hitachi ion sputter (E-1045, Hitachi Co., Tokyo, Japan), then observed with a Hitachi SU8010 FESEM (Hitachi Co., Tokyo, Japan) at the Public technology service center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Membrane sensitivity
Susceptibility testing was performed by the broth micro dilution method (CLSI 2012) with slight modification for erythromycin, rifampicin, deoxycholate or lysozyme. After lactose induction for 8, 12 or 24 h, E. coli BL21(DE3)/pX12345 or BL21(DE3)/pET28a cells were harvested by centrifuge for 5 min at 2200×g, then diluted to 1 × 105 colony forming units (CFU)/mL in LB broth. Aliquots of 100 μL of cells (~ 1×105 CFU/mL) were incubated with serial twofold dilutions of each test compound on 96-well plates at 35 °C in ambient air. Growth was measured spectrophotometrically (VersaMax Microplate Reader, Molecular Devices, Sunnyvale, CA, USA) as OD600 at ~ 16 h incubation. Each experiment was repeated three times and the data are represented as mean ± SE.
Enzyme assay
The β-xylosidase activity was determined by a colorimetric technique based on determining the amount of the product p-nitrophenol (pNP) released from the substrate p-nitrophenyl-β-d-xylopyranoside (pNPX) (Katapodis et al. 2006). The reaction mixture (50 mM phosphate (pH 7.0), 5 mM pNPX, an appropriate amount enzyme, total volume 0.25 mL) was incubated for 10 min at 55°C, and then the reaction was terminated by adding 0.75 mL of 2 M Na2CO3. The pNP produced was quantified by measuring the absorbance at 410 nm. One unit (U) of β-xylosidase activity was defined as the amount of enzyme producing 1 μmol of pNP per minute.
GenBank accession numbers
All the gene sequences used in the study are retrieved from the GenBank database, and their corresponding GenBank numbers were listed as follows: the P. thermophila J18 β-1,4-xylosidase gene PtXyl43, GU937001.1 (Teng et al. 2011); the codon-optimized PtXyl43 gene, MF818057; the Citrobacter sp. P (CGMCC No. 7536) chitinase gene CHIX, KC290945 (Xu et al. 2016); the hIL-2 gene hIL-2, AAA59140.1 (Robb 1984); the N-acyl homoserine lactonase (aiiO-AIO6) gene aiiO-AIO6 from Ochrobactrum sp. M231, JN572045 (Zhang et al. 2011).