Prefusion HCMV gB Structure Determination

Example 1

During the sample preparation the HCMV fusion inhibitor (compound 28 described in Bloom et al., Bioorganic & Medicinal Chemistry Letters 14 (2004) 3401-3406; see also FIG. 5D) was added to each step during the virus concentration, processing, extraction and purification to inhibit conversion of gB to the postfusion form.

Following crosslinking of the proteins on the virion surface with bis(sulfosuccinimidyl) glutarate (BS²G) and extraction of gB from the virion with detergent, the SM5-1 His/Strep-tagged Fab (Potzsch et al., PLoS pathogens 7(8):e1002172, 2011) was added to assist in purification and identification of gB by electron cryomicroscopy. The Fab-gB complexes were purified by an affinity column.

These extracted and purified proteins were then analyzed by electron cryomicroscopy for the presence of prefusion gB and used to solve the structure of a prefusion form.

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US11857622B2. Human cytomegalovirus GB polypeptide (2024-01-02). Pfizer Inc. [US]. Inventors: Yuhang Liu [US], Ye Che [US], Xiaoyuan Sherry Chi [US], Philip Ralph Dormitzer [US], Jennifer Anne Nicki [US], Xiaojie Yao [US].

Patent 2024

Detergent Electron cryomicroscopy Glutarate Hcmv Inhibit Isolation Pathogens Proteins Strain Virion Virus

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Variable analysis

independent variables

Addition of HCMV fusion inhibitor (compound 28) during virus concentration, processing, extraction, and purification
Crosslinking of proteins on the virion surface with bis(sulfosuccinimidyl) glutarate (BS^2G)
Extraction of gB from the virion with detergent
Addition of SM5-1 His/Strep-tagged Fab to assist in purification and identification of gB

dependent variables

Presence of prefusion gB
Structure of the prefusion form of gB

control variables

Not explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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