Cloning and Expression of Calpain Catalytic Core

The rat CAPN1 WT and p.V301W catalytic core sequences were cloned into a pUC57 vector with an XhoI restriction site, with an amino-terminal thrombin cleavage site and a carboxy-terminal TEV protease cleavage site followed by a 6xHis tag, as previously described (Moldoveanu et al. 2002 (link); Gakhar et al. 2016 (link)). Calpain constructs were transferred into the pMAL-C5X vector to obtain an amino-terminal maltose-binding protein (MBP) as a fusion partner. Sequence of the calpain-flanking regions was confirmed by sequencing of constructs. Plasmids were amplified and isolated from DH5α cells and then were transformed into Escherichia coli BL21 (DE3; New England Biolabs).

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Velez G., Bassuk A.G., Schaefer K.A., Brooks B., Gakhar L., Mahajan M., Kahn P., Tsang S.H., Ferguson P.J, & Mahajan V.B. (2018). A novel de novo CAPN5 mutation in a patient with inflammatory vitreoretinopathy, hearing loss, and developmental delay. Cold Spring Harbor Molecular Case Studies, 4(3), a002519.

Publication 2018

Escherichia coli BL21 (DE3;

Calpain Capn1 Catalytic core Cells Cleavage Escherichia coli Maltose binding protein Plasmids Tev protease Thrombin Vector

Corresponding Organization :

Other organizations : Stanford University, Smith-Kettlewell Eye Research Institute, University of Iowa, Palo Alto University, National Institutes of Health, National Eye Institute, New York University, Columbia University, Palo Alto Veterans Institute for Research

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Rat CAPN1 WT and p.V301W catalytic core sequences

dependent variables

Not explicitly mentioned

control variables

XhoI restriction site
Amino-terminal thrombin cleavage site
Carboxy-terminal TEV protease cleavage site
6xHis tag
Sequence of the calpain-flanking regions

controls

Positive control: Not mentioned
Negative control: Not mentioned

Annotations

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