Pet11a

Manufactured by Merck Group

Sourced in United States

PET11a is a laboratory equipment product manufactured by Merck Group. It is a plasmid vector used for the expression of recombinant proteins in Escherichia coli. The core function of PET11a is to serve as a cloning and expression vector for the production of target proteins in bacterial systems.

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Lab products found in correlation

Hek293, by American Type Culture Collection (1 mentions) Hepg2, by American Type Culture Collection (1 mentions) A549 cell line, by American Type Culture Collection (1 mentions) Bl21 de3, by Merck Group (1 mentions) Pegifnα 2a, by Roche (1 mentions)

6 protocols using pet11a

Cytokine Expression and Cell Line Acquisition

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Human IFN-α2a was purchased from PeproTech (Rocky Hill, NJ, USA; Cat. #: 300-02A) and PEG-IFN-α2a (PEGASYS) from Hoffman-La Roche Ltd. (Basel, Switzerland). Wild-type IFN-λ1 and IFN-λ analogs were expressed and prepared in-house. The prokaryotic expression vector pET11a, together with the competent cell BL21 (DE3), was purchased from EMD Millipore (Billerica, MA, USA). HepG2, HEK293, and A549 cell lines were purchased from ATCC (Manassas, VA, USA). Huh-7.5.1 cells were kindly provided by F Chisari (Scripps Research Institute, La Jolla, CA, USA). Sources of other materials and reagents are described in the relevant sections. No ethics statement was required from the Institutional Review Board of Jilin University for the use of these cell lines.

Yu D., Zhao M., Dong L., Zhao L., Zou M., Sun H., Zhang M., Liu H, & Zou Z. (2016). Design and evaluation of novel interferon lambda analogs with enhanced antiviral activity and improved drug attributes. Drug Design, Development and Therapy, 10, 163-182.

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Cloning and Expression of Nucleoporins

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Nup358^RBD4-CycH, Nup62^FL, and Nup153^CTD from Homo sapiens, as well as yeast nup Nsp1 from Saccharomyces cerevisiae, were cloned as 10 × His-MBP-SUMO-nup-SNAP constructs into a pET-28a-derived vector (Novagen) and expressed in BL21-Gold (DE3). Cells were grown in Terrific Broth at 37 °C in a shaking incubator until they reached an optical density at 600 nm (OD₆₀₀) of 1.0. Protein expression was then induced with 1 mM IPTG for 4 h at 20 °C before collection by centrifugation. Cell pellets were refrigerated at −80 °C until use. All CA constructs were cloned into pET-11a (EMD Millipore). CA proteins were overexpressed in BL21-Gold (DE3) cells at 25 °C for 12 h by induction with 0.5 mM IPTG at an OD₆₀₀ of 0.8. Cell pellets were collected by centrifugation and refrigerated at −80 °C until use.

González A., Rossini C., Eisner M, & Eisner T. (1999). Sexually transmitted chemical defense in a moth (Utetheisa ornatrix). Proceedings of the National Academy of Sciences of the United States of America, 96(10), 5570-5574.

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Recombinant Expression of Cor a 1 Allergens

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Synthetic genes coding for Cor a 1.0101; Cor a 1.0104 (Genescript, Piscataway, New Jersey, USA) and Cor a 1.0401 (optimized for codon-usage in E. coli) were cloned via NdeI, BamHI into the expression vector pET11a (Novagen-Merck, Germany). To obtain Cor a 1.0102 and Cor a 1.0103, plasmids pET11a Cor a 1.0104 and pET11a Cor a 1.0101, respectively, were used as templates for site-directed mutagenesis according to the QuickChange Method Cornell iGEM 2012” protocol (http://2012.igem.org/wiki/images/a/a5/Site_Directed_Mutagenesis.pdf).
Gene expression for all unlabelled, ¹⁵N, and ¹⁵N, ¹³C labelled allergens was performed as described previously for pET11a_Bet v 1a (Bet v 1.0101)^{33 (link)} using (¹⁵NH₄)₂SO₄ and ¹³C-glucose. An amino acid sequence alignment of the Cor a 1 and Bet v 1 proteins used in this study is shown in Supplementary Fig. S7. The protein bank entry accession numbers and a sequence identity matrix of the proteins are listed in Supplementary Table S4.

Jacob T., von Loetzen C.S., Reuter A., Lacher U., Schiller D., Schobert R., Mahler V., Vieths S., Rösch P., Schweimer K, & Wöhrl B.M. (2019). Identification of a natural ligand of the hazel allergen Cor a 1. Scientific Reports, 9, 8714.

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Recombinant Expression and Purification of Nucleoporins

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NUP153CTD from homo sapiens (amino acids 896-1475), as well as yeast nucleoporin NSP1 (amino acids 2-603) from S. cerevisiae, was cloned as 10× His-MBP-SUMO-NUP153 (or NSP1)-SNAP constructs (Figure S1) into a pET-28a-derived vector (Novagen) and expressed in BL21-Gold (DE3). Cells were grown in TB at 37 °C with shaking until they reached an OD 600 of 0.8. Protein expression was then induced with 1 mM IPTG for 3 hrs at 20 °C before collection by centrifugation. Cell pellets were refrigerated at -80 °C until use.
All CA constructs were cloned into pET-11a (EMD Millipore). CA-Foldon was generated by directly fusing Foldon to the C-terminus of CA. CA proteins were overexpressed in BL21-Gold (DE3) cells at 25 °C for 12 hr by induction with 0.5 mM IPTG at 0.8 OD 600 . Cell pellets were collected by centrifugation and refrigerated at -80 °C until use. Human CPSF6 was cloned into bacterial expression vector and expressed.

Shen Q., Xu C., Jang S., Xiong Q., Devarkar S.C., Tian T., Bedwell G.J., Tripler T.N., Hu Y., Yuan S., Temple J., Shi J., Aiken C., Engelman A., Perilla J.R., Lusk C.P., Lin C., & Xiong Y. (2020). A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid.

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Heterologous Expression of BglM-G1 in E. coli

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For the expression of BglM-G1 in E. coli, codon optimization of the gene sequence encoding BglM-G1 was carried out using the online tool JCat^{42 (link)}. The codon-optimized gene sequence was synthesized (GeneArt^®, Regensburg, Germany) with additional 5′ NdeI and 3′ EcoRI restriction sites. Digested bglM-G1 was then ligated into pOE, linearized with the same enzymes, for overexpression and purification via His-tag. pOE is a pET-11a (Merck Millipore, Billerica, MA, USA) derivative recently developed in our lab for this purpose^{43 (link)}. pOE contains an N-terminal 6× His-tag followed by a TEV-protease specific cleavage site. This added the additional N-terminal amino acid sequence MHHHHHHENLYFQGH to BglM-G1 during expression. After TEV cleavage, the scar peptide GH remained. The resultant plasmid pOEbglM-G1 was transformed into E. coli BL21 (DE3) for protein expression.

Mhaindarkar D., Gasper R., Lupilov N., Hofmann E, & Leichert L.I. (2018). Loss of a conserved salt bridge in bacterial glycosyl hydrolase BgIM-G1 improves substrate binding in temperate environments. Communications Biology, 1, 171.

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Strain Construction and Growth Conditions

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All E. coli strains used are listed in Table 2. Standard genetic methods including transformation and P1 vir transduction were used for strain construction.
Cells were grown in Luria-Bertani (LB; 1% tryptone, 0.5% yeast extract, 0.5% NaCl) medium at 30°C for ts strains under permissive conditions and 37°C (ftsA12) or 42°C (all others) under non-permissive conditions. All non-ts strains were grown at 37°C. Antibiotic concentrations were as previously described, and culture growth was monitored by optical density as previously reported (Haeusser et al., 2014 (link)) Detailed procedures in preparation for microscopic imaging are provided below.
Expression of cloned genes from vectors derived from pET11a (Novagen – EMD Millipore) was induced with 1 mM isopropyl-β-D-galactopyranoside (IPTG) (Fisher Scientific) and from pDSW-derived vectors with 0.5 mM IPTG. Gene expression from pKG-derived vectors (J.S. Parkinson, University of Utah) was induced with 0.1, 1.0, or 5.0 μM sodium salicylate (Mallinkrodt), as indicated in text and figure legends.
General DNA and protein manipulation and analysis, including spot titers, were performed as previously described (Haeusser et al., 2014 (link)).

Haeusser D.P., Rowlett V.W, & Margolin W. (2015). A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling. Molecular microbiology, 97(5), 988-1005.

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