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Pfuse

Manufactured by InvivoGen
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Sourced in United States, France

PFUSE is an automated device designed for the fusion of plasmid DNA. It provides a controlled and efficient method for the fusion of DNA fragments, enabling the assembly of complex genetic constructs. The core function of PFUSE is to facilitate the seamless combination of DNA sequences through a standardized and reproducible process.

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Lab products found in correlation

Expifectamine, by Thermo Fisher Scientific (3 mentions) Endotoxin removal kit, by Thermo Fisher Scientific (3 mentions) Expi293, by Thermo Fisher Scientific (3 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (3 mentions) Expifectamine kit, by Thermo Fisher Scientific (2 mentions) Protein a coated beads, by Thermo Fisher Scientific (1 mentions) Phlsec, by Addgene (1 mentions) Nickel nta resin, by Thermo Fisher Scientific (1 mentions) Pfuse higg1 fc, by InvivoGen (1 mentions) Pegfp, by Takara Bio (1 mentions) Pentr4 vector, by Thermo Fisher Scientific (1 mentions)

12 protocols using pfuse

1

Recombinant CLEC-2 and Podoplanin Protein Production

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For the recombinant human CLEC-2–6xHis tag and human podoplanin-rabbit Fc vector overexpression, the extracellular domain of the cDNA of human CLEC-2 and podoplanin were cloned into a pHLSEC (Addgene) and pFUSE (Invivogen) expression vectors. The constructs were used to transient transfection into human embryonic kidney HEK293T cells. Cells were grown in complete Dulbecco's modified eagle medium (DMEM; supplemented with 10% fetal bovine serum, glutamine 5%, and 100 U/mL of penicillin/streptomycin). Once 60% of cell confluence was achieved, the medium was exchanged for serum-free DMEM and transfected using the PEI method. Proteins secreted into the medium and were collected after 4 days. hCLEC-2–6xHis tag and h-podoplanin-rFc were purified using a Nickel-NTA Resin (Thermo Fisher) and protein A-coated beads (Thermo Fisher), respectively, in a gravity purification column. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was used to characterize proteins. Protein coding sequences of recombinant proteins can be found in the Supplementary Information.
Morán L.A., Di Y., Sowa M.A., Hermida-Nogueira L., Barrachina M.N., Martin E., Clark J.C., Mize T.H., Eble J.A., Moreira D., Pollitt A.Y., Loza M.I., Domínguez E., Watson S.P, & García Á. (2022). Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist. Thrombosis and Haemostasis, 122(8), 1361-1368.
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2

Recombinant Protein Production for COVID-19 Research

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Spike-RBD-monomer, Spike-RBD-Fc, Spike ectodomain (Secto), and ACE2-Fc were produced as biotinylated proteins as previously described.34 (link) VH were subcloned from the VH-phagemid into an E. coli expression vector pBL347. VH2 and VH3 were cloned into pBL347 with a 20-amino acid Gly-Ser linker. VH-Fc were cloned into a pFUSE (InvivoGen) vector with a human IgG1 Fc domain. All constructs were sequence verified by Sanger sequencing. VH, VH2, and VH3 constructs were expressed in E. coli C43(DE3) Pro + using an optimized autoinduction media and purified by protein A affinity chromatography similarly to Fabs (Supplementary Figure 11).30 (link) VH-Fc were expressed in Expi293 BirA cells using transient transfection (Expifectamine, Thermo Fisher Scientific). Four days after transfection, the media was harvested, and VH-Fc were purified using protein A affinity chromatography. All proteins were buffer exchanged into PBS by spin concentration and stored in aliquots at −80°C. The purity and integrity of proteins were assessed by SDS-PAGE. All proteins were endotoxin removed using an endotoxin removal kit (Thermo Fischer) prior to use in neutralization assays.
Bracken C.J., Lim S.A., Solomon P., Rettko N.J., Nguyen D.P., Zha B.S., Schaefer K., Byrnes J.R., Zhou J., Lui I., Liu J., Pance K., Zhou X.X., Leung K.K, & Wells J.A. (2020). Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2. Nature chemical biology, 17(1), 113-121.
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3

Recombinant Protein Production for COVID-19 Research

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Spike-RBD-monomer, Spike-RBD-Fc, Spike ectodomain (Secto), and ACE2-Fc were produced as biotinylated proteins as previously described.34 (link) VH were subcloned from the VH-phagemid into an E. coli expression vector pBL347. VH2 and VH3 were cloned into pBL347 with a 20-amino acid Gly-Ser linker. VH-Fc were cloned into a pFUSE (InvivoGen) vector with a human IgG1 Fc domain. All constructs were sequence verified by Sanger sequencing. VH, VH2, and VH3 constructs were expressed in E. coli C43(DE3) Pro + using an optimized autoinduction media and purified by protein A affinity chromatography similarly to Fabs (Supplementary Figure 11).30 (link) VH-Fc were expressed in Expi293 BirA cells using transient transfection (Expifectamine, Thermo Fisher Scientific). Four days after transfection, the media was harvested, and VH-Fc were purified using protein A affinity chromatography. All proteins were buffer exchanged into PBS by spin concentration and stored in aliquots at −80°C. The purity and integrity of proteins were assessed by SDS-PAGE. All proteins were endotoxin removed using an endotoxin removal kit (Thermo Fischer) prior to use in neutralization assays.
Bracken C.J., Lim S.A., Solomon P., Rettko N.J., Nguyen D.P., Zha B.S., Schaefer K., Byrnes J.R., Zhou J., Lui I., Liu J., Pance K., Zhou X.X., Leung K.K, & Wells J.A. (2020). Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2. Nature chemical biology, 17(1), 113-121.
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4

Production and Purification of RBD-Based Proteins

Cited 1 time
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Spike-RBD-monomer, Spike-RBD-Fc, Spike ectodomain (Secto), and ACE2-Fc were produced as biotinylated proteins as previously described.34 (link) VH were subcloned from the VH-phagemid into an E. coli expression vector pBL347. VH2 and VH3 were cloned into pBL347 with a 20-amino acid Gly-Ser linker. VH-Fc were cloned into a pFUSE (InvivoGen) vector with a human IgG1 Fc domain. All constructs were sequence verified by Sanger sequencing. VH, VH2, and VH3 constructs were expressed in E. coli C43(DE3) Pro + using an optimized autoinduction media and purified by protein A affinity chromatography similarly to Fabs.30 (link) VH-Fc were expressed in Expi293 BirA cells using transient transfection (Expifectamine, Thermo Fisher Scientific). 4 days after transfection, media was harvested, and VH-Fc were purified using protein A affinity chromatography. All proteins were buffer exchanged into PBS by spin concentration and stored in aliquots at −80°C. Purity and integrity of proteins were assessed by SDS-PAGE. All proteins were endotoxin removed using an endotoxin removal kit (Thermo Fischer) prior to use in neutralization assays.
Bracken C.J., Lim S.A., Solomon P., Rettko N.J., Nguyen D.P., Zha B.S., Schaefer K., Byrnes J.R., Zhou J., Lui I., Liu J., Pance K., Zhou X.X., Leung K.K, & Wells J.A. (2020). Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike. bioRxiv.
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5

Generation of Chimeric Antibodies for HA Targeting

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XY102, 6F12, KB2, 9H10, and 29E3 have been previously described (29 (link)– (link)32 (link), 34 (link)). Chimeric IgG and IgA antibodies were generated as previously described (36 (link)). Briefly, the variable regions of the group 1 HA stalk-binding monoclonal antibodies 6F12 and KB2 were cloned into human IgG and IgA backbones (pFUSE; InvivoGen). An identical strategy was used for 29E3, a strain-specific antibody that recognizes the head domain of A/California/04/09 HA.
Mullarkey C.E., Bailey M.J., Golubeva D.A., Tan G.S., Nachbagauer R., He W., Novakowski K.E., Bowdish D.M., Miller M.S, & Palese P. (2016). Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner. mBio, 7(5), e01624-16.
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6

Transient Protein Expression in Expi293 Cells

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All proteins were expressed and purified from Expi293 BirA cells according to established protocol from the manufacturer (Thermo Fisher Scientific). Briefly, 30 μg of pFUSE (InvivoGen) vector encoding the protein of interest was transiently transfected into 75 million Expi293 BirA cells using the Expifectamine kit (Thermo Fischer Scientific). For the IgG and Fab proteins, 15 μg of each chain was transfected. Enhancer was added 20 h after transfection. Cells were incubated for a total of 3 d at 37 °C in an 8% CO2 environment before the supernatants were harvested by centrifugation. Fc-fusion proteins were purified by Protein A affinity chromatography and His-tagged proteins were purified by Ni-NTA affinity chromatography. Purity and integrity were assessed by SDS/PAGE. Purified protein was buffer exchanged into PBS and stored at −80 °C in aliquots.
Elledge S.K., Zhou X.X., Byrnes J.R., Martinko A.J., Lui I., Pance K., Lim S.A., Glasgow J.E., Glasgow A.A., Turcios K., Iyer N., Torres L., Peluso M.J., Henrich T.J., Wang T.T., Tato C.M., Leung K.K., Greenhouse B, & Wells J.A. (2020). Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection. medRxiv.
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7

Bispecific Antibody Production in Expi293 Cells

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Expi293 (Life Technologies) cells were transiently co-transfected with two pFUSE (InvivoGen) vectors harboring either the Fab heavy chain and the Fab light chain genetically fused to the αCD19 scFv, or the Fab heavy chain fused to a human Fc and the Fab light chain, at a ratio of 1:1 for BiTE and IgG respectfully. The ExpiFectamine 293 transfection kit (Life Technologies) was used for transfections as per manufacturer’s instructions. Cells were incubated for 7 days at 37 ˚C in a 5% CO2 environment before the supernatants were harvested by centrifugation. Protein was purified by Protein A affinity chromatography and assessed for quality and integrity by SDS-PAGE.
Martinko A.J., Truillet C., Julien O., Diaz J.E., Horlbeck M.A., Whiteley G., Blonder J., Weissman J.S., Bandyopadhyay S., Evans M.J, & Wells J.A. (2018). Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins. eLife, 7, e31098.
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8

Transient Co-Transfection of Expi293 Cells

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Expi293 (Life Technologies) cells were transiently co-transfected with two pFUSE (InvivoGen) vectors harboring the AZ1 heavy chain and the AZ1 light chain genetically fused to the αCD19 scFv at a ratio of 1:1. The ExpiFectamine 293 transfection kit (Life Technologies) was used for transfections as per manufacturer’s instructions. Cells were incubated for 7 days at 37 °C in a 5% CO2 environment before the supernatants were harvested by centrifugation. Protein was purified by Protein A affinity chromatography and assessed for quality and integrity by SDS-PAGE (Supplementary Fig. 14).
Hill Z.B., Martinko A.J., Nguyen D.P, & Wells J.A. (2017). Human Antibody-Based Chemically Induced Dimerizers for Cell Therapeutic Applications. Nature chemical biology, 14(2), 112-117.
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9

Transient Co-Transfection of Expi293 Cells

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Expi293 (Life Technologies) cells were transiently co-transfected with two pFUSE (InvivoGen) vectors harboring the AZ1 heavy chain and the AZ1 light chain genetically fused to the αCD19 scFv at a ratio of 1:1. The ExpiFectamine 293 transfection kit (Life Technologies) was used for transfections as per manufacturer’s instructions. Cells were incubated for 7 days at 37 °C in a 5% CO2 environment before the supernatants were harvested by centrifugation. Protein was purified by Protein A affinity chromatography and assessed for quality and integrity by SDS-PAGE (Supplementary Fig. 14).
Hill Z.B., Martinko A.J., Nguyen D.P, & Wells J.A. (2017). Human Antibody-Based Chemically Induced Dimerizers for Cell Therapeutic Applications. Nature chemical biology, 14(2), 112-117.
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10

Engineered S. cerevisiae EBY100 Expression

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For the transformation of S. cerevisiae EBY100, the vector pYSDM2 used was adapted from pYSDM1 in [33 (link)]. Hereby, the order of the HA-tag was modified, placed next to the c-myc, and therefore a free amino terminus was created. The expression vector pFUSE-hIgG1-Fc (derived from pFUSE, InvivoGen (San Diego, CA, USA)) was used for the expression of scFv-Fc in Expi293F.
Fux A.C., Casonato Melo C., Schlahsa L., Burzan N.B., Felsberger A., Gessner I., Fauerbach J.A., Horejs-Hoeck J., Droste M, & Siewert C. (2024). Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin. International Journal of Molecular Sciences, 25(4), 2297.
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