Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Dan A. De Cotiis, Matthew P. Woll, Todd E. Fox, R. Blake Hill, Robert Levenson, John Flanagan

Research output: Contribution to journalArticle

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Abstract

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 6× 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 μM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50 mg of homogenous myristoylated NCS-1 is obtained from 1 L 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 ΔEF) 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.

Original languageEnglish (US)
Pages (from-to)103-112
Number of pages10
JournalProtein Expression and Purification
Volume61
Issue number2
DOIs
StatePublished - Oct 1 2008

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Escherichia coli
Neuronal Calcium-Sensor Proteins
Calcium
His-His-His-His-His-His
frequenin calcium sensor proteins
Exocytosis
Myristic Acid
Mutant Proteins
Protein C
GTP-Binding Proteins
Recombinant Proteins
Yeasts
Metals
Sodium
Growth

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

De Cotiis, Dan A. ; Woll, Matthew P. ; Fox, Todd E. ; Hill, R. Blake ; Levenson, Robert ; Flanagan, John. / Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli. In: Protein Expression and Purification. 2008 ; Vol. 61, No. 2. pp. 103-112.
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Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli. / De Cotiis, Dan A.; Woll, Matthew P.; Fox, Todd E.; Hill, R. Blake; Levenson, Robert; Flanagan, John.

In: Protein Expression and Purification, Vol. 61, No. 2, 01.10.2008, p. 103-112.

Research output: Contribution to journalArticle

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AU - Woll, Matthew P.

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AU - Flanagan, John

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N2 - 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 6× 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 μM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50 mg of homogenous myristoylated NCS-1 is obtained from 1 L 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 ΔEF) 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.

AB - 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 6× 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 μM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50 mg of homogenous myristoylated NCS-1 is obtained from 1 L 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 ΔEF) 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.

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