Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: Comparison with the native protein from bovine brain

Douglas C. Au, Elizabeth D. Apel, Edwin R. Chapman, Roger P. Estep, Teresa Nicolson, Daniel R. Storm

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities for calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.

Original languageEnglish (US)
Pages (from-to)8142-8148
Number of pages7
JournalBiochemistry
Volume28
Issue number20
StatePublished - 1989
Externally publishedYes

Fingerprint

GAP-43 Protein
Escherichia coli
Brain
Complementary DNA
Proteins
Calmodulin
Guanidine
Recombinant Proteins
Sepharose
Calmodulin-Binding Proteins
Escherichia coli Proteins
Post Translational Protein Processing
Sodium Dodecyl Sulfate
Protein Kinase C
Phospholipids
Animals
Plasmids
Cells
Availability
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli : Comparison with the native protein from bovine brain. / Au, Douglas C.; Apel, Elizabeth D.; Chapman, Edwin R.; Estep, Roger P.; Nicolson, Teresa; Storm, Daniel R.

In: Biochemistry, Vol. 28, No. 20, 1989, p. 8142-8148.

Research output: Contribution to journalArticle

Au, Douglas C. ; Apel, Elizabeth D. ; Chapman, Edwin R. ; Estep, Roger P. ; Nicolson, Teresa ; Storm, Daniel R. / Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli : Comparison with the native protein from bovine brain. In: Biochemistry. 1989 ; Vol. 28, No. 20. pp. 8142-8148.
@article{80ede307757b4d8895ea96c5b532fd40,
title = "Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: Comparison with the native protein from bovine brain",
abstract = "Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities for calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.",
author = "Au, {Douglas C.} and Apel, {Elizabeth D.} and Chapman, {Edwin R.} and Estep, {Roger P.} and Teresa Nicolson and Storm, {Daniel R.}",
year = "1989",
language = "English (US)",
volume = "28",
pages = "8142--8148",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "20",

}

TY - JOUR

T1 - Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli

T2 - Comparison with the native protein from bovine brain

AU - Au, Douglas C.

AU - Apel, Elizabeth D.

AU - Chapman, Edwin R.

AU - Estep, Roger P.

AU - Nicolson, Teresa

AU - Storm, Daniel R.

PY - 1989

Y1 - 1989

N2 - Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities for calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.

AB - Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities for calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.

UR - http://www.scopus.com/inward/record.url?scp=0024458692&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024458692&partnerID=8YFLogxK

M3 - Article

C2 - 2532540

AN - SCOPUS:0024458692

VL - 28

SP - 8142

EP - 8148

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 20

ER -