Pet28a

Manufactured by Takara Bio

Sourced in Japan, China, United States

The PET28a is a plasmid vector used for the expression and purification of recombinant proteins in E. coli. It contains a T7 promoter, a multiple cloning site, and a His-tag sequence for affinity purification.

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PET28a vector PET28a expression vector

20 protocols using pet28a

Cloning, Expression, and Antibody Production of GlPGM1 and GlPGM3 from G. lemaneiformis

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The ORFs of GlPGM1 and GlPGM3 were amplified, as mentioned above, and then cloned into the pMD18-T vector (Takara, Dalian, China) for sequencing (Youkang Biotech, Hangzhou, China). The GlPGM1 and GlPGM3 from the pMD18-T vector were then cut, using restriction endonuclease and cloned into expression vector pET-28a (+) (Takara, Dalian, China) to obtain pET-28a-GlPGM1 and pET-28a-GlPGM3 recombinant strains, respectively. The constructed plasmids were transformed into the expression strain E. coli BL21 (Takara, Dalian, China). The recombinant strains of GlPGM1and GlPGM3 were incubated and induced with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3–5 h at 28 °C. The overexpressed proteins GlPGM1and GlPGM3 were purified and checked by 12.5% SDS-PAGE. The E. coli with recombinant pET-28a without IPTG was used as the negative control.
The GlPGM1 and GlPGM3 polyclonal antibodies were made by Hangzhou Aiting Biological Technology Co., Ltd. (Hangzhou, China). The purified proteins, GlPGM1 and GlPGM3, were used as antigens to immunize rabbits for the production of the polyclonal antiserum. A Western blot analysis with the antibodies was conducted to check the expression of GlPGM1 and GlPGM3 from G. lemaneiformis (Figure S7, Supplementary Materials).

Chen Q., Yu X., Liu S., Luo S., Chen X., Xu N, & Sun X. (2022). Identification, Characteristics and Function of Phosphoglucomutase (PGM) in the Agar Biosynthesis and Carbon Flux in the Agarophyte Gracilariopsis lemaneiformis (Rhodophyta). Marine Drugs, 20(7), 442.

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Cloning and Mutagenesis of AM1_1557g2

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The nucleotide sequence of AM1_1557g2 was cloned into pET28a (Novagen) using the In-Fusion HD Cloning kit (TaKaRa). The DNA fragment corresponding to AM1_1557g2 was PCR amplified using the synthetic primers 5′-CGCGGCAGCCATATGTATGAGCGTAATATTGCT-3′ (forward primer) and 5′-CTCGAATTCGGATCCTCATGCTTCTGCTTTATCTCT-3′ (reverse primer), genomic DNA from A. marina MBIC11017, and PrimeSTAR Max DNA polymerase. pET28a was PCR amplified using the synthetic primers 5′-CATATGGCTGCCGCGCGG-3′ (forward primer) and 5′-GGATCCGAATTCGAGCTC-3′ (reverse primer), pET28a, and PrimeSTAR Max DNA polymerase. A plasmid expressing AM1_1557g2 (pET28a_AM1_1557g2) was then constructed with the TaKaRa in-fusion system reagents. pET28a_AM1_1557g2_C304A was generated using the primers 5′-AGAGACGCACATTTAGAGATTTTGGAA-3′ (forward primer) and 5′-TAAATGTGCGTCTCTATAAGATTCTTG-3′ (reverse primer), pET28a_AM1_1557g2, and PrimeSTAR Max Basal Mutagenesis kit reagents (TaKaRa). pET28a_AM1_1557g2_L337N was obtained in the same way as described above with a primer set (5′- CTATCAGAATAACGTCCCACGTCAATG-3′, 5′- ACGTTATTCTGATAGGCTGCCAGCAA-3′). The sequences of the genes encoding AM1_1557g2, C304A and L337N were verified by DNA sequencing.

Narikawa R., Nakajima T., Aono Y., Fushimi K., Enomoto G., N.i.-.N.i.-.W.i.n., Itoh S., Sato M, & Ikeuchi M. (2015). A biliverdin-binding cyanobacteriochrome from the chlorophyll d–bearing cyanobacterium Acaryochloris marina. Scientific Reports, 5, 7950.

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Cloning and Expression of VP3 Protein in E. coli

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A VP3 sequence (GenBank accession number ABI52864.1) was codon-optimized for expression in E. coli and purchased from GenScript (New Jersey, USA). The VP3 gene was subcloned into the 6×His tag expression vector pET28a (Novagen) using BamH I and Xho I restriction endonucleases. Overlapping gene fragments varying in size (150 or 390 bp) and covering the full-length VP3 sequence were PCR-amplified (Fig. 1). The larger VP3 gene fragments (FA, FM and FP, 390 bp) were inserted into pET28a, whereas the shorter VP3 fragments (F1 to F7, 150 bp) were inserted into pET28a-GFP, between EcoR I and Xho I restriction sites. The pET28a-GFP was constructed by inserting the green fluorescent protein gene, which was PCR-amplified from pEGFP-C3 (Clontech Laboratories, CA, USA), into the BamH I site on pET28a (upstream of the EcoR I and Xho I sites). The E. coli DH5α strain was used to propagate the plasmids. The oligonucleotides used in this work are shown in Additional file 6. All constructed plasmids were sequenced to verify the absence of mutations and the correct in-frame insertion of amplicons.

Palka A.P., Assunção de Matos T.R., de Souza C., Eugênio D.S., Krieger M.A., Fragoso S.P, & Pavoni D.P. (2021). Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil. BMC Veterinary Research, 17, 259.

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Cloning and Transformation of Tyr p 13 Protein

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The recombinant pMD20-T-Tyr p 13 plasmid was digested with NdeI and XhoI to release the Tyr p 13 cDNA. The cDNA was separated by agarose gel electrophoresis and purified from the gel using the Agarose Gel DNA Purification kit v2.0. The DNA Ligation kit (cat. no. D6023; Takara Biotechnology Co., Ltd.) was used to sub-clone the cDNA into the expression vector pET28a(+) (cat. no. N72770; Novagen; Merck KGaA) to create pET28a(+)-Tyr p 13. E. coli DH5α cells (cat. no. D9057; Takara Biotechnology Co., Ltd.) were transformed with pET28a(+)-Tyr p 13 plasmids. Positive clones were selected by blue/white screening on LB plates containing 50 µg/ml kanamycin and verified by restriction enzyme analysis with NdeI and XhoI.

Wang N., Zhou Y., Wu M., Zhu H, & Cui Y. (2020). Expression, epitope prediction and IgE-binding of the Tyrophagus putrescentiae group 13 allergen. Experimental and Therapeutic Medicine, 20(5), 21.

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Recombinant scFv Protein Purification

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The genes encoding e23sFv-derived scFvs fused with His-tag at the 3' end and with 5'- and 3'-flanking NcoI and NotI restriction sites were cloned into the prokaryotic expression vector pET28a (Takara Biotechnology Co., Ltd.). The resulting recombinant plasmids were verified by agarose gel electrophoresis using 1% gel and ethidium bromide visualization. The recombinant plasmids were then transformed into E. coli BL21 (DE3) (Takara Biotechnology Co., Ltd.), and after 3 h of induction with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37˚C, the bacteria were harvested and sonicated for 30 min at 2 sec intervals on ice for use in SDS-PAGE analysis to demonstrate the presence of the proteins of interest in inclusion bodies. For the purification of inclusion bodies, the precipitates were dissolved in 8 M urea buffer and subjected to Ni²⁺-NTA affinity chromatography using Ni-NTA His•Bind Resins (Novagen; MilliporeSigma) according to the manufacturer's instructions. After extensive washing with 100 mM imidazole buffer, the bound proteins were eluted at room temperature with 1,000 mM imidazole buffer and refolded by linearly decreasing the urea gradient (7, 6, 4, 2, 1 and 0 M). The renatured proteins were finally dialyzed into PBS (pH 7.4) and quantified by BCA assay (Pierce; Thermo Fisher Scientific, Inc.).

Ou-Yang Q., Ren J.L., Yan B., Feng J.N., Yang A.G, & Zhao J. (2020). Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv. Experimental and Therapeutic Medicine, 21(2), 136.

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Cultivation and Expression of Saccharomonospora viridis

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Saccharomonospora viridis DSM 43017 was obtained from the China General Microbiological Culture Collection Center, Beijing (reference number CGMCC 4.1324). The strain was cultivated in a shaker flask at 45°C in STS medium (1.0% (w/v) soy peptone, 1.0% (w/v) glucose, 0.2% (w/v) yeast extract, 0.2% (w/v) NaCl, and 0.2% casein enzymatic hydrolysate, all from Biodee, Beijing, China), adjusted pH to 8.0 with NaOH prior to autoclaving.
Escherichia coli DH5α and E. coli BL21 (Tiangen, Beijing, China) (were cultivated in Luria Bertani (LB) medium at 37°C for gene cloning, sequencing, and expression. The pEASY-T3 vector (TransGen, Beijing, China) was used for plasmid gene cloning and sequencing. The plasmid pET-28a (+) (Takara Bio, Otsu, Japan) was used as an expression vector. Manganese peroxidase (MnP), azure B, brilliant green, reactive blue 19, reactive green 19, reactive yellow 2, reactive black 5, reactive red 120, malachite green and crystal violet were purchased from Sigma (St. Louis, MO, USA). All other reagents used were of analytical grade unless otherwise stated.

Yu W., Liu W., Huang H., Zheng F., Wang X., Wu Y., Li K., Xie X, & Jin Y. (2014). Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp. PLoS ONE, 9(10), e110319.

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Recombinant Expression and Purification of H9-HA2 Protein

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The HA2 gene of AH/BRI99/16 (H9N2) was amplified by RT-PCR using the gene specific primers. Then the fragment was amplified with the specific program as follows: 5 min at 95 °C for predenaturation; 35 cycles of 30 s at 95 °C for denaturation, 30 s at 54 °C for annealing, 1 min at 72 °C for elongation; and finally, 10 min at 72 °C for overall elongation. The obtained HA2 fragment was subcloned into the prokaryotic expression vector pET-28a (Takara, Japan). The recombinant plasmid was verified by Sanger sequencing and then transformed into E. coli BL21 cells for the expression of HA2 protein. The expression of H9-HA2 protein including His tag was induced with 1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG), and the protein was purified using a Ni-NTA agarose (Thermo, USA) according to the manufacturer’s instructions. The purified fusion protein was identified with SDS-PAGE and Western blot.

Huang J., Huang N., Fan M., Zhao L., Luo Y., Ding P., Tian M., Liu Q., Guo Y., Zhao J., Zheng Y., Zhang H, & Ping J. (2020). Hemagglutinin stalk-based monoclonal antibody elicits broadly reactivity against group 1 influenza A virus. Virology Journal, 17, 191.

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Cloning β-glucosidase from C. bescii

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Genomic DNA from C. bescii DSM 6725 (DSMZ, Braunschweig, Germany), E. coli ER2566 (New England Biolabs, Hertfordshire, UK), and pET-28a(+) (Takara, Shiga, Japan) were used as the DNA template source of the β-glucosidase gene (GenBank accession number ACM59590), host cells, and expression vector, respectively [16 (link)]. The β-glucosidase gene from C. bescii DSM 6725 was cloned using the one-step isothermal assembly method as described previously.

Kil T.G., Kang S.H., Kim T.H., Shin K.C, & Oh D.K. (2019). Enzymatic Biotransformation of Balloon Flower Root Saponins into Bioactive Platycodin D by Deglucosylation with Caldicellulosiruptor bescii β-Glucosidase. International Journal of Molecular Sciences, 20(16), 3854.

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Recombinant Tar Periplasmic Domain Expression

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A plasmid, pET-Tar, that expresses the periplasmic domain, residues 26–193 (TAR-E), of E. coli Tar was constructed using the expression vector pET-28a(+) (Novagen). This construct encodes an N-terminal methionine, a six-His tag and thrombin-recognition and enterokinase-recognition sites, followed by a Tar periplasmic sequence encompassing Gly26–Gln193. A DNA fragment encoding the periplasmic domain of Tar was amplified by PCR from E. coli DH5α genomic DNA using the forward primer 5′-ATG GCT AGC GAT GAC GAC GAC AAG GGC AGC CTG TTT TTT TCT TC-3′ (NheI site in bold) and the reverse primer 5′-CTC GAA TTC TCA TTA TTG CCA CTG GGC AAA TC-3′ (EcoRI site in bold). The resulting PCR product was digested with NheI and EcoRI, and was cloned into pET-28a(+) using a DNA ligation kit (Takara Bio, Tokyo, Japan). This construct was termed pET-Tar.

Mise T., Matsunami H., Samatey F.A, & Maruyama I.N. (2014). Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate. Acta Crystallographica. Section F, Structural Biology Communications, 70(Pt 9), 1219-1223.

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Recombinant Protein Expression System

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The sequence of YtnP was amplified by PCR from the genomic DNA of DH82 using the primerytnP-F (5′-GGAATTCATGGAGACATTGAATATTGGGAATATTTC-3′) and ytnP-R (5′-CGGGATCCTTATTTTTTCTCCCGTTTGACAGATG-3′) were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and was digested by the restriction enzymes BamHI and EcoRI, and subsequently ligated the multiple cloning sites into pET28a with the T4 ligase (Takara, China). The engineered expression clone, which was driven by T7 promoter, and framed with 6× Histidine (His) at both N- and C-terminal of target enzyme, was transferred in E. coli BL21 for protein expression.
The operon of LuxR-P_luxI-lacO-RFP was provided by Xiamen University (Xiamen, China), and was conducted on pET28a between the restriction sites of NdeI and HpaI. The re-engineered reporter plasmid was then transferred in E. coli BL21 (named LuxR- RFP for short) to construct reporter strain for rapid detection of AHLs.

Sun X., Hill P., Liu J., Qian J., Ma Y, & Zhou S. (2021). Marine-Source Quorum Quenching Enzyme YtnP to Improve Hygiene Quality in Dental Units. Marine Drugs, 19(4), 225.

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