Cloning and expression of chicken skeletal muscle troponin I in <i>Escherichia coli</i>: The role of rare codons on the expression level

Quaggio, Ronaldo Bento; Ferro, Jesus Aparecido; Monteiro, P. B.; Reinach, Fernando C.

doi:10.1002/pro.5560020618

Cited by 29 publications

(30 citation statements)

References 28 publications

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“…2). Troponin I, which accumulates in inclusion granules and causes the plasmid to become unstable (Quaggio et al, 1993), remained soluble when coexpressed with troponin C, as expected if troponin I and troponin C were interacting.…”

Section: Resultssupporting

confidence: 64%

“…Vectors designed to express chicken skeletal muscle troponin C, troponin I or troponin T-3 (PET-TnC, PET-TnI and PET-TnT) as single subunits have been constructed in our laboratory (Quaggio et al, 1993;Da Silva et al, 1993;Farah et al, 1994). These vectors were used to construct plasmids capable of coexpressing the different troponin subunits.…”

Section: Plasmidsmentioning

confidence: 99%

“…When expressed in E. coli, troponin C is found in the soluble fraction after cell lysis (Reinach and Karlsson, 1988). Troponin I is a basic (PI = 9.3), highly insoluble protein (Johnson et al, 1980), found in inclusion bodies when expressed in E. coli (Quaggio et al, 1993). Troponin T-3 isoform contains a high percentage of charged residues (Smillie et al, 1988) but has a close to neutral PI (PI = 6.5).…”

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confidence: 99%

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Assembly of functional skeletal muscle troponin complex in Escherichia coli

Malnic

Reinach

1994

European Journal of Biochemistry

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The production of multi-subunit proteins of eukaryotic origin in Escherichia coli usually relies on the different subunits being expressed individually and the protein being reassembled in vitro. Here we describe the construction and characterization of plasmids capable of coexpressing the three subunits of chicken skeletal muscle troponin complex in E. coli. We demonstrate that the troponin subunits assembled in the cytoplasm of E. coli cell are fully functional. The troponin complex was purified to homogeneity in high yields. When reconstituted into actin filaments, the complex assembled in vivo was capable of regulating the myosin ATPase with a calcium dependence that was identical to the complex reconstituted in vitro. These results demonstrate that the coexpression of the subunits of a protein complex can prevent the accumulation of denatured proteins in inclusion granules.

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Section: Resultssupporting

confidence: 64%

Section: Plasmidsmentioning

confidence: 99%

mentioning

confidence: 99%

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Assembly of functional skeletal muscle troponin complex in Escherichia coli

Malnic

Reinach

1994

European Journal of Biochemistry

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“…The C79S and C96S mutants were then introduced into these constructs as for pET3a⅐cTnC"WT." The fsTnI construct in pET3a was a gift from F. C. Reinach (19). Sequence analysis of this construct, which expressed poorly in our experiments, showed that the codons for Arg-13 and Arg-14 were both CGG (rare in E. coli).…”

Section: Methodsmentioning

confidence: 86%

Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

Dargis

Pearlstone

Barrette-Ng³

et al. 2002

Journal of Biological Chemistry

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“…The recombinant tropomyosins ASTm and nfTm were expressed and purified as described (19) Recombinant chicken fast skeletal troponin I, troponin C, and TnT were expressed and purified separately, and the troponin complex was reconstituted as described (39,40). Tropomyosin and troponin concentration were determined according to the method described by Hartree (41) using ASTm as a standard (⑀ 278 nm 1% ϭ 2.9 (19)).…”

Section: Construction Of Vectors For Bacterial Expression Of Tropomyomentioning

confidence: 99%

Quantitative Analysis of Tropomyosin Linear Polymerization Equilibrium as a Function of Ionic Strength

Sousa¹,

Farah

2002

Journal of Biological Chemistry

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Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken ␣-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin headto-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mM (between I ‫؍‬ 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin.Skeletal muscle tropomyosin (Tm) 1 is a 284-residue dimeric in-register coiled-coil protein that plays a central role in the regulation of muscle contraction through its interactions with actin and troponin (Tn) in the thin filament (1-5). Analysis of its primary structure revealed the existence of an extensive and almost perfect heptad repeat (abcdefg) in which hydrophobic residues at positions a and d create a dimerization interface that stabilizes the coiled-coil structure (6, 7). Further analysis of tropomyosin sequences also uncovered seven 39.5-residue pseudo-repeats, which may reflect the tropomyosin 7:1 binding stoichiometry with actin (8). Also associated with both tropomyosin and actin is the troponin complex, which consists of the Ca 2ϩ binding subunit (troponin C), the inhibitory subunit (troponin I), and the tropomyosin binding subunit (TnT). Tn-Tm interactions occur mainly between the C-terminal half of TnT and the region near position 190 of Tm and between the Nterminal half of TnT and the C-terminal of the Tm molecule (for review, see Refs. 9 -13).Muscle tropomyosin high viscosity at low ionic strength was noted from the time of its first isolation (14). Soon thereafter Tsao et al. (15) describe the ionic strength dependence of the viscosity in more detail and Kay and Bailey (16) relate changes in the polymer length with salt concentration and propose that polymerization is due to simple end-to-end or head-to-tail aggregation, which was later confirmed by in-depth studies of cardiac Tm via sedimentation velocity, sedimentation equilibrium, osmometry, viscometry, and optical rotatory dispersion (17).Although muscle Tm is acetylated at its N terminus, recombinant non-fusion Tm (nfTm) expressed in bacteria lacks this modification and does not polymerize or bind to actin (18,19). Recombinant Tm expressed in insect cells (20) or yeast (21) has its N terminus acetylated and is functional. Because the Nacetylated initiation methionine in the native protein occupies an internal position in the coiled-coil struc...

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Cloning and expression of chicken skeletal muscle troponin I in Escherichia coli: The role of rare codons on the expression level

Cited by 29 publications

References 28 publications

Assembly of functional skeletal muscle troponin complex in Escherichia coli

Assembly of functional skeletal muscle troponin complex in Escherichia coli

Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

Quantitative Analysis of Tropomyosin Linear Polymerization Equilibrium as a Function of Ionic Strength

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