Xylanase genes were cloned using PCR with synthesized cDNA as the template and primers listed in Additional file 1 : Table S2. PCR products were purified and cloned into a pLYJ-163 vector (xynA, xynB, xynC, xynD) and pEASY-Blunt E1 vector (xynE) to construct recombinant plasmids, which were transformed into E. coli DH5α cells and confirmed by DNA sequencing (TSINGKE, Qingdao, China). The correct recombinant plasmids were transformed into E. coli BL21 (DE3) for protein expression. When the absorbance at 600 nm (OD600) reached 0.6–0.8, strains were induced using 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG, Solarbio, Beijing, China) for 20 h at 16 °C. Cells were centrifuged and resuspended in buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). After ultrasonic fragmentation, xylanases were purified using HisCap Co 6FF resin (Smart-life Sciences, Changzhou, China). The eluent was replaced with optimal buffer (50 mM Na2HPO4, 20 mM citrate, pH 5.0) by ultrafiltration (3 kDa membrane, Millipore, Billerica, MA) at 4 °C. Pure enzymes were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined using the Bradford method [44 (link)].
Isopropyl β d thiogalactopyranoside iptg
Manufactured by Solarbio
Sourced in China
Isopropyl-β-D-thiogalactopyranoside (IPTG) is a chemical compound commonly used in molecular biology as an inducer for gene expression. It is a synthetic analog of lactose and serves as a powerful tool in the regulation of gene expression in bacterial systems.
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Lab products found in correlation
Kanamycin, by Solarbio (5 mentions)
3 kda membrane, by Merck Group (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ni2 sepharose high performance, by GE Healthcare (1 mentions)
Ni nta superflow sepharose column, by Merck Group (1 mentions)
Microplate analyzer, by Bio-Rad (1 mentions)
Histrap ff crude, by GE Healthcare (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Clonexpression 2 one step cloning kit, by Vazyme (1 mentions)
Dpni, by Takara Bio (1 mentions)
γ linolenic acid gla, by Merck Group (1 mentions)
Sypro orange protein gel stain, by Merck Group (1 mentions)
Mut express 2 fast mutagenesis kit v2, by Vazyme (1 mentions)
E coli bl21 de3, by Solarbio (1 mentions)
Modified bradford protein assay kit, by Sangon (1 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (1 mentions)
High affinity ni nta column, by GenScript (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
13 protocols using isopropyl β d thiogalactopyranoside iptg
1
Cloning and Expression of Xylanase Genes
2
PLGA Nanoparticle Synthesis and Characterization
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PLGA polymer (the ratio of lactide: glycolide feed was 50:50, Mw: 10 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd., Shandong, China. The Ni2+ Sepharose High Performance was purchased from GE Healthcare Life Sciences (Piscataway, NJ). The isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Solarbio Life Sciences (Beijing, China). The oleic acid-albumin-dextrose-catalase (OADC) enrichment solution was from BD Biosciences (New York, NY, USA), and 7H9 and 7H10 Middlebrook media were from Difco (New York, NY, USA). The mouse monoclonal His-tag antibody was purchased from Proteintech (Wuhan, Hubei, China). Mouse TNF-α, Il-1β, IFN-γ, IgG, and IgA ELISA (enzyme-linked immunosorbent assay) kits were purchased from Neobioscience Technology (Shenzhen, Guangdong, China). The micro-BCA protein assay kit was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).
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PLGA polymer (the ratio of lactide: glycolide feed was 50:50, Mw: 10 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd., Shandong, China. The Ni2+ Sepharose High Performance was purchased from GE Healthcare Life Sciences (Piscataway, NJ). The isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Solarbio Life Sciences (Beijing, China). The oleic acid-albumin-dextrose-catalase (OADC) enrichment solution was from BD Biosciences (New York, NY, USA), and 7H9 and 7H10 Middlebrook media were from Difco (New York, NY, USA). The mouse monoclonal His-tag antibody was purchased from Proteintech (Wuhan, Hubei, China). Mouse TNF-α, Il-1β, IFN-γ, IgG, and IgA ELISA (enzyme-linked immunosorbent assay) kits were purchased from Neobioscience Technology (Shenzhen, Guangdong, China). The micro-BCA protein assay kit was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).
3
Recombinant RBD Protein Production
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The SARS-CoV-2 S-RBD protein was purchased from Novamab (Shanghai, China). The SARS-CoV RBD protein was purchased from Biorbyt (Cambridge, UK). The MERS-CoV RBD protein was purchased from prospecbio (Ness Ziona, Israel). The SARS-CoV-2 N protein was purchased from Fapon Biotech (Dongguan, China). Freund’s incomplete adjuvant, horseradish peroxidase (HRP)-conjugated anti-HA monoclonal antibodies (mAbs), anti-mouse IgG-alkaline phosphatase (ALP), Bis (p-nitrophenyl) phosphate (BNPP), and Ni-NTA superflow sepharose column were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-HA tag antibody was obtained from Abcam (Cambridge, UK). The E. coli TG1 and WK6 competent cells were kept in our laboratory. Anti-SARS-CoV-2 S-RBD mAb (No.bs-41407R) was purchased from Bioss (Beijing, China). 96-well plates were purchased from Merck Corning (Darmstadt, Germany). The SARS-CoV-2 S protein RBD (Omicron), ampicillin (Amp), tetramethylbenzidine (TMB), and isopropyl β-D-thiogalactopyranoside (IPTG) were from Solarbio Biotechnology (Beijing, China).
4
Serum ELISA for Antibody and Cytokine Quantification
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Serum was extracted from the blood samples (50 μL) for ELISA analysis. The PRV-gB-specific antibody level was evaluated by the ID.vet commercial ELISA kit (France). The levels of mouse IL-2, IL-4, IFN-γ, TNF-α, and IL-6 were evaluated by a Neobioscience commercial ELISA kit (China). For the evaluation of SVA-specific VP2 antibodies, an ELISA method was established. The SVA-VP2 gene fragment was inserted into PET32a (Novagen) and transformed into the BL21(DE3) Escherichia coli strain. The recombinant protein was induced with 0.5 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG; Solarbio), and the strain was cultured for 48 h at 16°C and 100 rpm. The optimal antigen coating concentration was 62.5 ng/mL, and the serum was diluted 1:200. After incubation for 1.5 h at 37°C, a secondary antibody (1:5,000 diluted HRP-conjugated goat anti-mouse IgG) was added and incubated for 1 h at 37°C. The TMB substrate chromodeveloping solution (Solarbio) was colored for 15 min, followed by the addition of 2 M H2SO4 to each well to terminate the reaction. The absorbance value was detected at an opical density at 450 nm (OD450) using a microplate analyzer (Bio-Rad) within 15 min.
5
Purification of His-Tagged Proteins from E. coli
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Escherichia coli BL21 (DE3) cells were cultured in LB medium supplemented with 50 μg/mL kanamycin at 37 °C until the optical density of the medium reached 0.6–0.8 at 600 nm. Subsequently, for protein induction, isopropyl-β-D-thiogalactopyranoside (IPTG; Solarbio, Beijing, China) was added to the medium at the final concentration of 50 mM. The culture was incubated for 20 h in a shaker (20 °C, 200 rpm). After centrifugation, the precipitate was harvested and resuspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Ni2+ affinity chromatography (HisTrap™ FF crude; GE Healthcare, Buckinghamshire, UK) was used for protein purification after ultrasonic fragmentation. The eluent was replaced with PC buffer (20 mM sodium phosphate, 10 mM citrate, pH 6.0) and subjected to ultrafiltration through a 3-kDa molecular cutoff membrane (Millipore, Billerica, MA, USA) at 4 °C. SDS-PAGE was performed on a 12% (w/v) polyacrylamide gel, and the protein bands were stained with Coomassie Brilliant Blue R-250 (Sigma–Aldrich, St. Louis, MO, USA). Protein concentration was determined using the classical Bradford method [47 (link)].
6
Recombinant L-Asparaginase Production
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The recombinant plasmid pET-30a(+)-BLASNase harboring type II L-asparaginase gene (ansA) from Bacillus licheniformis and E. coli BL21 (DE3) strain were preserved in our laboratory. The gene was PCR-amplified using the 2× Hieff Canace® PCR Master Mix from Yeasen Biotech (Shanghai, China). The DNA cloning kit and ClonExpression® II One Step Cloning kit were obtained from Vazyme (Nanjing, China). DpnI was acquired from Takara Biotechnology (Dalian, China). The Bradford Protein Assay Kit was obtained from Beyotime (Shanghai, China). Primers were synthesized from GenScript (Nanjing, China). Solarbio (Beijing, China) provided isopropyl β-d -thiogalactopyranoside (IPTG) and Kanamycin. L-Asparagine, trichloroacetic acid, and all other reagents were from Aladdin (Shanghai, China). All chemicals were analytical grade.
7
Cloning and Expression of Lox Gene from E. norvegicus
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The LOX coding gene from E. norvegicus DSM 15893 (accession number WP_074927588) was codon-optimized and cloned to the plasmid pET-28a (+) with Nde I and Xho I, as the inserted restriction sites by Genewiz. E. coli strains DH5α (Novozymes, Wilmington, USA) and BL21 (DE3) (Novozymes, Wilmington, USA) were preserved in our laboratory. Kanamycin and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Solarbio (Beijing, China). PUFA standards, including linoleic acid (LA), α-linolenic acid (ALA), and γ-linolenic acid (GLA), were purchased from Sigma (Steinheim, Germany). Hydroxy fatty acid (HFA) standards, including 13-hydroxyoctadecaenoic acid (13-HODE), 13(S)-hydroxyoctadeca trienoic acid (13S-HOTrE), were purchased from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were analytical grade. The study design was shown in Figure 1 .
8
Cloning and Expression of L-Asparaginase
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The plasmid pET30a-AsA harboring the L-asparaginase gene from A. soli was preserved in our laboratory (GenBank accession number: OK019339). The screening and construction of the plasmid pET30a-AsA were previously reported [20 (link)]. The expression strain E. coli BL21 (DE3) was purchased from Solarbio (Beijing, China). Mut Express® II Fast Mutagenesis Kit V2 and FastPure Gel DNA Extraction Mini Kit for mutant construction were purchased from Vazyme Biotech (Nanjing, China). The primers were synthesized by GenScript (Nanjing, China). Kanamycin and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Solarbio (Beijing, China). Modified Bradford Protein Assay Kit was purchased from Sangon Biotech (Shanghai, China). SYPRO® Orange Protein Gel Stain was purchased from Sigma-Aldrich (St. Louis, USA). Enzyme substrates and all other reagents used in this research were purchased from Aladdin (Shanghai, China).
9
Expression and Purification of Recombinant dIL-2
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The dIL-2 gene with a full-length of 434 bp (GenBank: JX239765.1 ) was synthesized by GenScript Biotech Co., Ltd. (Nanjing, China) and then cloned into the pET-28a expression vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) cells and then was induced by 1 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG, Solarbio, Beijing, China) at 37°C for 6 h. The protein containing His-tag was purified using a high-affinity Ni-NTA column (GenScript USA Inc., Nanjing, China) and carried out with endotoxin removal twice. The presence of expressed recombinant proteins was evaluated by 12% SDS-PAGE then recognized by Western blotting. Purified dIL-2 protein was determined by bioactivity assay of recombinant human interleukin-2 in Veterinary Pharmacopoeia of the People's Republic of China. The PET protein (1 mg/mL, the tag protein of the pET-28a vector was expressed and purified in our lab, with endotoxin at a concentration of less than 10 EU/mg) was used as control for dIL-2.
10
Recombinant Protein Expression in E. coli
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The plasmid pET-28a from Invitrogen (Carlsbad, CA, USA) was adopted as the expression vector in Escherichia coli DH5α and BL21 (DE3) were utilized to clone and expression of hosts, separately. The recombinant strains of E. coli were cultivated in LB medium (0.5% w/v yeast extract, 1% w/v tryptone, and 1% w/v NaCl). Phusion DNA polymerase, restriction endonuclease, and T4 DNA ligase were purchased from ThermoFisher Scientific (Shanghai, China). The plasmid extraction kit, PCR product purification kit, and isopropyl-β-D-thioga-lactopyranoside (IPTG) were purchased from Solarbio (Beijing, China). Nickel columns and nickel resin used for affinity chromatography were purchased from GE Health-care (Uppsala, Sweden). Substrate FB1 (purity of 98%) was obtained from Pribolab Ltd. (Qingdao, China, http://wwwpribolab.com/ , accessed on 4 March 2022) and dissolved in acetonitrile-water. All additional reagents and chemicals are of analytical grade.
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