Pet303 expression vectors

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PET303 expression vectors are a series of plasmid vectors designed for the expression of recombinant proteins in Escherichia coli. They feature a T7 promoter system for high-level protein expression and a variety of antibiotic resistance markers for selection.

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Expression vectors (2 citations)

3 protocols using pet303 expression vectors

Recombinant Protein Expression and Purification

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The DNA encoding the protein sequences were synthesized and cloned into pET303 expression vectors by Thermo Fisher Scientific (Waltham, MA, USA), including a N-terminal methionine and a C-terminal polyhistidine tag with sequence GGGGSHHHHHH. The proteins were overexpressed in E. coli bacteria (BL21(DE3)) and purified following a two-step chromatography protocol, adapted from the method described previously [22] (link). Specific details of the protein production protocol are described in the Supplementary Material. Protein purity (>95 %) was assessed by SDS-PAGE and the identity of each protein was confirmed by mass spectrometry.
Synthetic peptide V39E derived from the S2 HR2 sequence was acquired from Genecust (Luxembourg), with a purity >95 %. The peptide (residues 1164–1202) was C-terminally tagged with a SGGY sequence to confer UV absorption at 280 nm, as well as N-acetylated and C-amidated. Protein and peptide concentrations were measured by UV absorption measurements at 280 nm with extinction coefficients calculated according to their respective amino acid sequences with the Expasy ProtParam server (https://web.expasy.org/protparam/) [49] (link).

Cano-Muñoz M., Polo-Megías D., Cámara-Artigas A., Gavira J.A., López-Rodríguez M.J., Laumond G., Schmidt S., Demiselle J., Bahram S., Moog C, & Conejero-Lara F. (2022). Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity. International Journal of Biological Macromolecules, 222, 2467-2478.

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Purification and Characterization of Mutant Proteins

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The DNA encoding protein sequences were synthesized and cloned into pET303 expression vectors by Thermo Fisher Scientific (Waltham, Massachusetts, MA, USA). To facilitate purification by Ni-Sepharose affinity chromatography, the protein sequences included a C-terminal histidine tag with sequence GGGGSHHHHHH. The sequences are shown in Table S1. Each triple mutant was named as covNHR followed by the identity of first mutation, i.e., covNHR-Q13I, covNHRQ23I, and covNHR-T30I. The proteins were overexpressed in E. coli and purified, as described previously [22 (link)]. The protein purity was assessed by SDS-PAGE, and the identity of each protein variant was confirmed by mass spectrometry analysis.
Synthetic CHR peptides (Table S1), both N-acetylated and C-amidated, were acquired from Genecust (Mondorf-les-Bains, Luxembourg), with a purity >95%. Protein and peptide concentrations were measured by UV absorption measurements at 280 nm with extinction coefficients calculated according to their respective amino acid sequences with the ExPasy ProtParam server (https://web.expasy.org/protparam/, accessed on 3 March 2020) [29 (link)].

Cano-Muñoz M., Cesaro S., Morel B., Lucas J., Moog C, & Conejero-Lara F. (2021). Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity. Biomolecules, 11(4), 566.

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Purification and Characterization of HIV-1 gp41 Proteins

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The NHR and CHR gp41 sequences used in this work are described in Figure S1. The reference gp41 sequence was taken from the full gp160 precursor glycoprotein of the HIV-1 BRU isolate (Swiss-Prot entry sp|P03377|ENV_HV1BR). CovNHR miniproteins were computationally designed using YASARA software. The DNA encoding the protein sequences were synthesized and cloned into pET303 expression vectors (Thermo Fisher Scientific, Waltham, USA). To facilitate purification by Ni- Sepharose affinity chromatography, the protein sequences were histidine tagged at the C terminus with the sequence GGGGSHHHHHH. The covNHR proteins were produced and purified following the protocol previously described [21 (link)]. Synthetic CHR peptides, both N-acetylated and C- amidated, were acquired from Genecust (Luxembourg), with a purity >95%. Protein and peptide concentrations were determined by UV absorption measurements at 280 nm using the extinction coefficients calculated according to their respective amino acid sequences with the ExPasy ProtParam server (https://web.expasy.org/protparam/ accessed on 2 February 2022) [46 (link)].

Cano-Muñoz M., Lucas J., Lin L.Y., Cesaro S., Moog C, & Conejero-Lara F. (2022). Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion. International Journal of Molecular Sciences, 23(5), 2794.

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