| Autor: |
De Cotiis DA; Department of Biochemistry and Molecular Biology, Penn State College of Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17033, USA., Woll MP, Fox TE, Hill RB, Levenson R, Flanagan JM |
| Jazyk: |
angličtina |
| Zdroj: |
Protein expression and purification [Protein Expr Purif] 2008 Oct; Vol. 61 (2), pp. 103-12. Date of Electronic Publication: 2008 Jun 25. |
| DOI: |
10.1016/j.pep.2008.06.006 |
| Abstrakt: |
We have developed a protocol to produce large quantities of high purity myristoylated and non-myristoylated neuronal calcium sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium-dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6x His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100-200 microM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50mg of homogenous myristoylated NCS-1 is obtained from 1L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 DeltaEF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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