Recombinant Human ERBB2 protein, hFc-tagged

• Cat.No.: ERBB2-41H
• Product Overview: Recombinant Human ERBB2 protein(P04626-1)(Thr23-Thr652), fused with C-terminal hFc tag, was expressed in HEK293.

Specification

• Species: Human
• Source: HEK293
• Tag: Fc
• Protein Length: Thr23-Thr652
• Tag: C-hFc
• Form: Lyophilized from 0.22μm filtered solution in PBS (pH 7.4). Normally 8% trehalose is added as protectant before lyophilization.
• Bio-activity: Immobilized Human Her2, hFc Tag at 5μg/ml (100μl/well) on the plate. Dose response curve for Biotinylated Anti-Her2 Antibody , hFc Tag with the EC50 of 0.20μg/ml determined by ELISA.
• Molecular Mass: The protein has a predicted MW of 96.1 kDa. Due to glycosylation, the protein migrates to 100-120 kDa based on Tris-Bis PAGE result.
• Endotoxin: Less than 1EU per μg by the LAL method.
• Purity: > 95% as determined by Tris-Bis PAGE; > 95% as determined by HPLC
• Storage: Reconstituted protein stable at -80°C for 12 months, 4°C for 1 week. Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
• Reconstitution: Centrifuge tubes before opening. Reconstituting to a concentration more than 100 μg/ml is recommended. Dissolve the lyophilized protein in distilled water.

Gene Information

• Gene Name: ERBB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) [ Homo sapiens ]
• Official Symbol: ERBB2
• Synonyms: ERBB2; v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); NGL, v erb b2 avian erythroblastic leukemia viral oncogene homolog 2 (neuro/glioblastoma derived oncogene homolog); receptor tyrosine-protein kinase erbB-2; CD340; HER 2; HER2; NEU; herstatin; p185erbB2; proto-oncogene Neu; c-erb B2/neu protein; proto-oncogene c-ErbB-2; metastatic lymph node gene 19 protein; tyrosine kinase-type cell surface receptor HER2; neuroblastoma/glioblastoma derived oncogene homolog; v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (neuro/glioblastoma derived oncogene homolog); NGL; TKR1; HER-2; MLN 19; HER-2/neu
• Gene ID: 2064
• mRNA Refseq: NM_001005862
• Protein Refseq: NP_001005862
• MIM: 164870
• UniProt ID: P04626

Citation

In vitro Fab display: a cell-free system for IgG discovery

Journal: Protein Engineering, Design and Selection    PubMed ID: 24586053    Data: 2014/4/1

Authors: Ryan L. Stafford, Marissa L. Matsumoto, Aaron K. Sato

Article Snippet:The coupled transcription/translation reactions were allowed to proceed for 60 min at 30°C.The coupled transcription/translation reactions were allowed to proceed for 60 min at 30°C.. The remaining selection was performed using standard protocols ( ) with 14 nM HER2/ErbB2 ECD (Creative BioMart) previously biotinylated.. Reverse transcription (RT)-PCR was performed using a one-step Transcriptor RT-PCR kit (Roche) with T7B/TolAk primers.Reverse transcription (RT)-PCR was performed using a one-step Transcriptor RT-PCR kit (Roche) with T7B/TolAk primers.

Conceptual overview of Fab display. ( a ) To display functional heterodimeric Fab domains from ribosomes two DNA templates are required for transcription: 1. A ‘Display Template’ lacking a stop codon at the end of a disordered TolA spacer, and 2. An ‘Expression Template’ with a stop codon. ( b ) A mixture of a display template and its complementary expression template co-expressed in a cell-free translation mixture enables the formation of a ternary complex of the functional Fab, ribosome and mRNA complex. For HC display, the HC display template is co-expressed with the LC expression template which enables the selection of a HC library. Alternatively, co-expression of the LC display template with the HC expression template enables LC display. ( c ) Co-expression of both the trastuzumab Fab HC and LC display and expression templates in either the HC or LC display format is required for significant recovery of the template following selection with Her2/ErbB2 ECD. This is consistent with the assembly of functional heterodimeric Fabs on stalled ribosomes as the models in (b) suggest. A reproducible, weak band is observable in the HC Display format when HC and LC are expressed in the absence of HER2/ErbB2 selection. This is presumably due to non-specific binding of the Fab-HC mRNA-ribosome complex. Similarly, a weak band is observable in the LC Display format when the LC only is displayed due to non-specific binding.

Screening and expression of IgGs with selected HCs. ( a ) IgGs from the selection were expressed on small scale (30 μl) in cell-free extract and screened by ELISA to find leads that were specific for CEA over HER2/ErbB2, streptavidin and a PBS control. Positive controls show that an anti-CEA scFv-Fc is specific for CEA and the trastuzumab IgG is specific for HER2/ErbB2. Experiments performed on separate days are presented in separate charts. ( b ) A non-reducing SDS–PAGE gel shows significant expression of each selected IgG at the correct molecular weight for the fully assembled product. Proteins are labeled with 14 C-Leu. ( c ) A reducing SDS–PAGE gel shows good expression of the majority of the reformatted complete HC and LC. Proteins are labeled with 14 C-Leu. A 14 C-labeled protein ladder (in kDa) is shown on the left lanes of the gels in (b) and (c).

Characterization of purified IgGs. ( a ) The final purified anti-CEA IgGs run predominantly as a single band on a non-reducing SDS–PAGE gel consistent with the full-length IgG. Under reducing conditions, bands consistent with the HC and LC fragments are observed. ( b ) Caliper analysis reveals the high purity of the final IgGs. The inset shows the same sample traces where the amount of protein is indicated by the band intensity. ( c ) An ELISA of the purified anti-CEA IgGs have sub-nanomolar EC 50 values for CEA. ( d ) A sample Biacore trace (red lines) for the 2-G3 anti-CEA IgG and global fit to a bivalent binding model (black lines). Binding was measured in duplicate at 1.3, 2.5, 5.1, 10.1, 20.3 and 40.5 nM over three separate channels. A bivalent model fit the data better than simple 1 : 1 binding as expected for a bivalent IgG. ( e ) The reformatted IgGs show apparent high affinities on MKN45 cells which express high levels of CEA, while the wt trastuzumab (expressed in CHO) IgG does not. ( f ) The wt trastuzumab IgG (expressed in CHO) binds to the ErbB2/HER2 expressing SKBR3 cells, but the selected CEA-specific IgGs do not.

Q&As

Do you have a recommended protocol for immobilizing any avi-tagged recombinant proteins on a streptavidin coated plate?

1.Prepare the streptavidin-coated plate by washing it with PBS (phosphate-buffered saline) or another suitable buffer to remove any impurities. 2.Dilute the Avi-tagged protein in the desired buffer at the desired concentration. It is important to use a buffer that is compatible with the protein and does not interfere with its activity or stability. 3.Add the diluted protein to the streptavidin-coated plate and incubate it for a suitable amount of time at room temperature or 4°C. The exact conditions will depend on the specific protein and the desired level of immobilization. 4.Wash the plate with a suitable buffer to remove any unbound protein. 5.Block the remaining binding sites on the plate with a suitable blocking agent such as BSA (bovine serum albumin) or casein. 6.Wash the plate again to remove any unbound blocking agent. 7.Use the immobilized protein for further experiments such as ELISA, Western blotting, or other assays.