Shrimp alkaline phosphatase
| Shrimp alkaline phosphatase | ||
|---|---|---|
| Mass / length primary structure | 475 amino acids , 52,971 Da | |
| Secondary to quaternary structure | Homodimer | |
| Cofactor | Magnesium, zinc | |
| Identifier | ||
| Gene name (s) | sap | |
| External IDs |
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| Enzyme classification | ||
| EC, category | 3.1.3.1. , Phosphatase | |
| Substrate | Phosphoric acid monoester and water | |
| Products | Alcohol and phosphate | |
| Occurrence | ||
| Parent taxon | Pandalus borealis | |
The Shrimp Alkaline Phosphatase ( SAP ) is a from Arctic shrimp ( Pandalus borealis isolated) enzyme , which in molecular biology is often used. As an alkaline phosphatase , it is indispensable for the animal in several metabolic pathways , for example in the dephosphorylation of the 5'-phosphate of DNA and RNA . The catalysis of this reaction step by the SAP is also used in the laboratory, with this special phosphatase having the advantage that it is completely deactivated by heating it for 20 minutes at 65 ° C, which keeps the reaction under control.
use
One of the most common areas of application of the SAP is in the cloning of genes or other DNA segments in plasmids : The circular vector (the plasmid) is cut with a restriction enzyme and thus linearized. The desired gene, which in the optimal case has been cut with the same enzymes , is added to the linearized vector . Ideally, the ends of the vector will attach to those of the inserted DNA double strand and a ligase will join the new plasmid.
If blunt ends or sticky ends with the same overlapping sequence occur during the linearization of the vector , the two ends of the vector can religate and thus reduce the yield of newly assembled plasmid - to prevent this, the 5 'ends of the vector are dephosphorylated with the SAP .
Alternatives for cloning are Calf Intestine Phosphatase and Antarctic Phosphatase .
Web links
- Shrimp Alkaline Phosphatase from Thermo Fisher Scientific
- Shrimp Alcaline Phosphatase at New England Biolabs
literature
- Sambrook, J .; Maniatis, T .; Russel, DW: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press; 3rd edition (2001) ISBN 0-87969-577-3
Individual evidence
- ↑ Cornel Mülhardt: The experimenter molecular biology / genomics . 7th edition. Springer-Verlag, Berlin / Heidelberg 2013, ISBN 978-3-642-34636-1 , p. 135 , doi : 10.1007 / 978-3-642-34636-1 .