14-IBSUlt-S11L

Highly reactive ultrapure Influenza B Virus, strain Singapore, recombinant nucleoprotein (IBV-rNP) from S. cerevisiae, lyophilized

Description: Influenza B virus (IBV) strain B/Singapore/222/79 full-length (560 a.a.) recombinant Nucleoprotein (UniProtKB/Swiss-Prot sequence accession number: P04666). Expressed in S. cerevisiae and purified from yeast lysates by combination of chromatographic techniques and ultracentrifugation in CsCl gradients under native (non-denaturing) conditions to ~97 % purity. This protein product is highly reactive in immunoassays.

Form: Lyophilized in PBS.

Storage: Shipped at ambient temperature (shipment costs: 65 € to Europe and 100 € to US). Lyophilized products may be stored at 2-8°C or at -20°C for longer periods.

Reconstitution: add 100 μl of deionized water for reconstitution of 100 µg of IBV-rNP in each separate vial. Check lyophilized material to be completely dissolved before use.

Concentration of reconstituted product: 1.0 mg/ml in PBS.

Purity: ~97% (including both major and minor isoforms) as determined by SDS-PAGE.

SDS-PAGE analysis showing IBV-rNP product 14-IBSUlt-S11L at approximately 62 kDa (2, 3 and 10 μg/lane, respectively).

Note on molecular weight and processing of IBV-rNP in yeast: Major IBV-rNP form is detected in SDS-PAGE as intact protein at ~62 kDa (which approximately corresponds to theoretically calculated MW from amino acid sequence ~61.5 kDa), but there is also minor slightly truncated isoform just below the major band. According to ESI-MS (electrospray ionization mass spectrometry) analysis, the major form of yeast-expressed IBV-rNP is N-terminally acetylated 2-560 aa protein, which is also found in native virus (see Hutchinson et al, PLoS Pathog. 2012;8(11):e1002993, Table 3). The minor form of purified protein represents acetylated form of 4-560 aa according to ESI-MS of 14-IBSUlt-S11L product sample. Therefore, for up to ~10 % of IBV-rNP molecules yeast starts translation from 4th codon ATG of aa Methionine instead of the first Met codon ATG (as for the major protein form). In the major IBV-rNP form 1st methionine is excised and the 2nd aa is acetylated (like in native IBV-NP), whereas after start from the 4th codon Met is not excised, but is acetylated itself.

Applications (tested): highly reactive in ELISA and other immunoassays, ESI-MS, Western blot, SDS-PAGE.

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