Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts.
2004
Abstract The two best known calpains, μ- and m-calpain, are Ca 2+ -dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca 2+ -linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (μ-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine–Dalgarno ribosome binding site. The results provide a simple method to obtain ∼10 mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
22
References
6
Citations
NaN
KQI