Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts.
The two best known calpains, micro- 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 (micro-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 approximately 10mg 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.
Larsen
Anna K
AK
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, 3, DK-1958 Frederiksberg C, Denmark.
De Veyra
Teresa
T
Jia
Zongchao
Z
Wells
Alan
A
Dutt
Previn
P
Elce
John S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D002154:Calpain; C449000:m-calpain