An Expression System of Rat Calmodulin Using T7 Phage Promoter inEscherichia coli

doi:10.1006/prep.1997.0807

Protein Expression and Purification

Volume 12, Issue 1, February 1998, Pages 25-28

https://doi.org/10.1006/prep.1997.0807 Get rights and content

Abstract

An efficient expression system of rat calmodulin inEscherichia coliis presented. To express rat calmodulin cDNA, we employed a pET expression vector which contains the T7 phage promoter and terminator. After transformation ofE. coliBL21(DE3) strain which carries T7 phage RNA polymerase inducible with isopropyl-β-d-thiogalactopyranoside, induction of the expression, and chromatography of soluble proteins on a phenyl–Sepharose column, about 250 mg of recombinant rat calmodulin was obtained from 1 liter ofE. coliculture. The recombinant calmodulin lacked the N-terminal methionine, and posttranslational modifications such as N^α-acetylation and methylation. This system facilitates the large amount preparation of calmodulin and the mutant proteins required for the structural analysis by NMR spectrometry and/or X-ray crystallography.

References (24)

R. Fischer et al.
Multiple divergent mRNAs code for a single human calmodulin
J. Biol. Chem.
(1988)
B. SenGupta et al.
Molecular analysis of human and rat calmodulin complementary DNA clones: Evidence for additional active genes in these species
J. Biol. Chem.
(1987)
D.M. Watterson et al.
The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain
J. Biol. Chem.
(1980)
J.R. Dedman et al.
Sequence homology of the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis with other Ca2+-binding proteins
J. Biol. Chem.
(1978)
H. Nojima et al.
Structure of a gene for rat calmodulin
J. Mol. Biol.
(1987)
H. Nojima
Structural organization of multiple rat calmodulin genes
J. Mol. Biol.
(1989)
J.A. Putkey et al.
Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene
J. Biol. Chem.
(1985)
R. Gopalakrishna et al.
Ca2+-induced hydrophobic site on calmodulin: Application for purification of calmodulin by phenyl–Sepharose affinity chromatography
Biochem. Biophys. Res. Commun.
(1982)
H. Taniguchi et al.
A mass spectrometric study on the in vivo posttranslational modification of GAP-43
J. Biol. Chem.
(1994)
H. Taniguchi et al.
Myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, is an in vivo substrate of proline-directed protein kinase(s): A mass spectroscopic analysis of the post-translational modifications
J. Biol. Chem.
(1994)

K. Yagi et al.

Identification of an activator protein for myosin light chain kinase as the Ca²⁺

J. Biol. Chem.

(1978)

Cited by (136)

Transcriptional, biochemical, and immunohistochemical analyses of CaMKKβ/2 splice variants that co-localize with CaMKIV in spermatids
2024, Cell Calcium
Ca²⁺/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream protein kinases, including CaMKI, CaMKIV, PKB/Akt, and AMPK; thus, regulates various Ca²⁺-dependent physiological and pathophysiological pathways. Further, CaMKKβ/2 in mammalian species comprises multiple alternatively spliced variants; however, their functional differences or redundancy remain unclear. In this study, we aimed to characterize mouse CaMKKβ/2 splice variants (CaMKKβ-3 and β-3x). RT-PCR analyses revealed that mouse CaMKKβ-1, consisting of 17 exons, was predominantly expressed in the brain; whereas, mouse CaMKKβ-3 and β-3x, lacking exon 16 and exons 14/16, respectively, were primarily expressed in peripheral tissues. At the protein level, the CaMKKβ-3 or β-3x variants showed high expression levels in mouse cerebrum and testes. This was consistent with the localization of CaMKKβ-3/-3x in spermatids in seminiferous tubules, but not the localization of CaMKKβ-1. We also observed the co-localization of CaMKKβ-3/-3x with a target kinase, CaMKIV, in elongating spermatids. Biochemical characterization further revealed that CaMKKβ-3 exhibited Ca²⁺/CaM-induced kinase activity similar to CaMKKβ-1. Conversely, we noted that CaMKKβ-3x impaired Ca²⁺/CaM-binding ability, but exhibited significantly weak autonomous activity (approximately 500-fold lower than CaMKKβ-1 or β-3) due to the absence of C-terminal of the catalytic domain and a putative residue (Ile478) responsible for the kinase autoinhibition. Nevertheless, CaMKKβ-3x showed the ability to phosphorylate downstream kinases, including CaMKIα, CaMKIV, and AMPKα in transfected cells comparable to CaMKKβ-1 and β-3. Collectively, CaMKKβ-3/-3x were identified as functionally active and could be bona fide CaMKIV-kinases in testes involved in the activation of the CaMKIV cascade in spermatids, resulting in the regulation of spermiogenesis.
Rapid detection of calmodulin/target interaction via the proximity biotinylation method
2023, Biochemical and Biophysical Research Communications
Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca²⁺-dependent biotinylation of a target protein kinase (Ca²⁺/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca²⁺-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.
Oligomerization of Ca<sup>2+</sup>/calmodulin-dependent protein kinase kinase
2022, Biochemical and Biophysical Research Communications
Ca²⁺/calmodulin-dependent protein kinase kinases (CaMKKα and β) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His–CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG–CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST–CaMKKα/His–CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca²⁺/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG–CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.
Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo
2020, Biochemical and Biophysical Research Communications
Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator- and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron-like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca²⁺/calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210.
Grb7-derived calmodulin-binding peptides inhibit proliferation, migration and invasiveness of tumor cells while they enhance attachment to the substrate
2020, Heliyon
The growth factor receptor bound protein 7 (Grb7) is a Ca²⁺-dependent calmodulin (CaM)-binding adaptor protein implicated, among other functions, in cell proliferation, migration and tumor-associated angiogenesis. The goal of this study was to determine whether a peptide based on the CaM binding site of Grb7 disrupts cellular processes, relevant for the malignancy of tumor cells, in which this adaptor protein is implicated. We designed synthetic myristoylated and non-myristoylated peptides corresponding to the CaM-binding domain of human Grb7 with the sequence ²⁴³RKLWKRFFCFLRRS²⁵⁶ and a variant peptide with the mutated sequence RKLERFFCFLRRE (W246E-ΔK247-S256E). The two non-myristoylated peptides bind dansyl-CaM with higher efficiency in the presence than in the absence of Ca²⁺ and they enter into the cell, as tested with 5(6)-carboxytetramethylrhodamine (TAMRA)-labeled peptides. The myristoylated and non-myristoylated peptides inhibit the proliferation, migration and invasiveness of A431 tumor cells while they enhance their adhesion to the substrate. The myristoylated peptides have stronger inhibitory effect than the non-myristoylated counterparts, in agreement with their expected higher cell-permeant capacity. The myristoylated and non-myristoylated W246E-ΔK247-S256E mutant peptide has a lesser inhibitory effect on cell proliferation as compared to the wild-type peptide. We also demonstrated that the myristoylated peptides were more efficient than the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibiting cell migration and equally efficient inhibiting cell proliferation.
Analyzing protein-ligand and protein-interface interactions using high pressure
2019, Biophysical Chemistry
Citation Excerpt :
Furthermore, the huge conformational change upon ligand binding is a clear marker of the binding state, and the strong negative net charge at pH = 7 facilitates adsorption at surfaces via electrostatic interactions. Finally, CaM can be produced as the wild type and as various mutants by gene expression in an efficient way [33]. A broad range of spectroscopic and scattering techniques can be applied to investigate protein interactions under high pressures.
All protein function is based on interactions with the environment. Proteins can bind molecules for their transport, their catalytic conversion, or for signal transduction. They can bind to each other, and they adsorb at interfaces, such as lipid membranes or material surfaces. An experimental characterization is needed to understand the underlying mechanisms, but also to make use of proteins in biotechnology or biomedicine. When protein interactions are studied under high pressure, volume changes are revealed that directly describe spatial contributions to these interactions. Moreover, the strength of protein interactions with ligands or interfaces can be tuned in a smooth way by pressure modulation, which can be utilized in the design of drugs and bio-responsive interfaces. In this short review, selected studies of protein-ligand and protein-interface interactions are presented that were carried out under high pressure. Furthermore, a perspective on bio-responsive interfaces is given where protein-ligand binding is applied to create functional interfacial structures.

View all citing articles on Scopus

¹: To whom correspondence should be addressed at Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Dengakugakubo, Kutukake-chou, Toyoake, Aichi 470-11, Japan. Fax: +81-562-93-8832. E-mail:[email protected].

View full text

Regular ArticleAn Expression System of Rat Calmodulin Using T7 Phage Promoter inEscherichia coli

Abstract

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Regular Article
An Expression System of Rat Calmodulin Using T7 Phage Promoter inEscherichia coli