Overexpression of Human Cardiac Troponin in Escherichia coli: Its Purification and Characterization

Lohmann, Karin; Westerdorf, Barbara; Maytum, Robin; Geeves, Michael A.; Jaquet, Kornélia

doi:10.1006/prep.2000.1328

Cited by 9 publications

(12 citation statements)

References 40 publications

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“…3. The results are similar to those published previously (3,4,15), showing a 3-4-fold reduction in k obs for the skeletal system in the absence of calcium and a 2-3-fold reduction for the cardiac system (13,28,30). Table I shows the K B values determined from these rates, which are therefore similar (0.5 for skeletal and 0.7 for cardiac) but with a small reproducible difference between them.…”

Section: Isolation Of Proteins From Bovine Heart and Rabbit Skeletal supporting

confidence: 89%

“…Dephosphorylation of TnI by PP2A was confirmed by measuring incorporation of 32 P by protein kinase A into dephosphorylated thin filaments (28). Fig.…”

Section: Isolation Of Proteins From Bovine Heart and Rabbit Skeletal mentioning

confidence: 95%

“…This material was used for reconstitution of thin filaments. Dephosphorylated cTn thin filaments were obtained by treatment of phosphorylated filaments with PP2A as this has been shown previously (28) to produce better regulating filaments.…”

Section: Isolation Of Proteins From Bovine Heart and Rabbit Skeletal mentioning

confidence: 99%

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Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

Maytum¹,

Westerdorf²,

Jaquet³

et al. 2003

Journal of Biological Chemistry

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There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K T (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca 2؉ -bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the ؉Ca 2؉ state of the two systems is similar whereas the ؊Ca 2؉ state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.

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Section: Isolation Of Proteins From Bovine Heart and Rabbit Skeletal supporting

confidence: 89%

“…Dephosphorylation of TnI by PP2A was confirmed by measuring incorporation of 32 P by protein kinase A into dephosphorylated thin filaments (28). Fig.…”

Section: Isolation Of Proteins From Bovine Heart and Rabbit Skeletal mentioning

confidence: 95%

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Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

Maytum¹,

Westerdorf²,

Jaquet³

et al. 2003

Journal of Biological Chemistry

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“…Similar to that seen in previous studies [20][21][22], the improvement of cardiac TnI expression by using the TnC-TnI bi-cistronic vector was moderate ( Table 1). The high level expression of TnC indicates the presence of an abundant amount of the bi-cistronic mRNA in the bacterial cell and the co-expression with cardiac TnI did not affect the translational efficiency and accumulation of human cardiac TnC protein in E. coli.…”

Section: Bi-cistronic Co-expression With Tnc Improves the Expression supporting

confidence: 87%

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

Jin

2007

Archives of Biochemistry and Biophysics

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Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca 2+ regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in E. coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5'-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5' but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation. KeywordsTroponin I; Protein conformation; Bi-cistronic expression; Codon usage Muscle contraction is regulated by intracellular Ca 2+ via the thin filament-based troponintropomyosin system. Troponin is a striated muscle-specific protein complex contains three subunits: the Ca 2+ -binding subunit troponin C (TnC), the tropomyosin binding subunit troponin T (TnT) and the inhibitory subunit troponin I (TnI) [1,2]. During muscle contraction, cytoplasmic Ca 2+ rises and binds to TnC to induce a series of conformational changes in the troponin complex that release the inhibition of actomyosin ATPase and activate force development [3]. Three homologous TnI genes (cardiac, fast skeletal muscle, and slow skeletal muscle) have evolved in vertebrates to encode muscle-type-specific TnI isoforms [4]. Primary structures of cardiac, fast and slow skeletal muscle TnI isoforms are highly conserved. The main structural difference is a cardiac TnI-specific ~30 amino acids NH 2 -terminal extension [5].* To whom correspondence should be addressed. Tel: (847) 570-1960. Fax: (847)570-1865. E-mail: jpjin@northwestern.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered whi...

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“…The cDNAs encoding human cardiac troponin C (hcTnC) and the major isoform of hcTnT were derived from clone TC1 [15] and from clone HCTNT2 [16], respectively. The cDNA for hcTnC was subcloned into the pET3c vector [13] and the one for hcTnT into the pSBETc vector [17,18] thus serving as templates for the generation of the hcTnC domains (hcTnC‐N‐DOM and hcTnC‐C‐DOM) and the C‐terminal domain of hcTnT (hcTnT‐C‐DOM), respectively. The primers for the generation of the hcTnC‐N‐DOM construct were 5′‐GAA TTC ATA TGG ATG ACA TCT ACA AGG CTG‐3′ (sense) and 5′‐AAG CTT GGA TCC CTA AGA TTT CCC TTT GCT GTC GTC C‐3′ (antisense); those for the hcTnC‐C‐DOM construct were 5′‐GAA TTC ATA TGG TTC GGT GCA TGA AGG ACG‐3′ (sense) and 5′‐AAG CTT GGA TCC CTA CTC CAC ACC CTT CAT GAA CTC‐3′ (antisense) and those for the hcTnT‐C‐DOM construct were 5′‐GCG CGA ACA TAT GGG GGG TTA CAT CCA GAA GCA G‐3′ (sense) and 5′‐TGA GGA TCC TCA TTT CCA GCG CCC GGT GAC TT‐3′ (antisense).…”

Section: Methodsmentioning

confidence: 99%

The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A ³¹P‐NMR study

2002

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Cardiac troponin I, the inhibitory subunit of the heterotrimeric cardiac troponin (cTn) complex is phosphorylated by protein kinase A at two serine residues located in its heartspecific N-terminal extension. This flexible arm interacts at different sites within cTn dependent on its phosphorylation degree. Bisphosphorylation is known to induce conformational changes within cTnI which finally lead to a reduction of the calcium affinity of cTnC. However, as we show here, the bisphosphorylated cTnI arm does not interact with cTnC, but with cTnT and/or cTnI. ß

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Overexpression of Human Cardiac Troponin in Escherichia coli: Its Purification and Characterization

Cited by 9 publications

References 40 publications

Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A ³¹P‐NMR study

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Overexpression of Human Cardiac Troponin in Escherichia coli: Its Purification and Characterization

Cited by 9 publications

References 40 publications

Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A 31P‐NMR study

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The interaction of the bisphosphorylated N‐terminal arm of cardiac troponin I‐A ³¹P‐NMR study