Expression of spinach plastocyanin in <i>E. coli</i>

Nordling, Margareta; Olausson, T.; Lundberg, Lennart

doi:10.1016/0014-5793(90)80517-m

Cited by 30 publications

(14 citation statements)

References 29 publications

(25 reference statements)

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“…In this paper, however, correct maturation of Synechocystis PC in E. coli is demonstrated by comparing the N-end amino acid sequence of the expressed protein with that of PC synthesized in the cyanobacterium. A more complicated procedure for overexpression of spinach PC in E. coliwhich is based on the use of an expression vector containing the signal peptide sequence of Pseudomonas aeruginosa azurin -has been developed by Nordling et al [24]. The N-terminal amino acid sequence of Synechocystis PC (ANATVKMG) was compared with those of the copper-proteins from other cyanobacteria such as Microcystis aeruginosa (ETFTVKMG) and Anabaena variabilis (ETYTVKLG).…”

Section: Resultsmentioning

confidence: 99%

Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E. coli transformed cells are identical

Hervás¹,

Navarro²,

Navarro³

et al. 1993

FEBS Letters

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Native plastocyaninfrom Synechocystis 6803 has been isolated and punfied to electrophoretic homogeneity. The corresponding gene @etE) has been cloned and expressed in E. coli, thus leading to a protein completely identical to plastocyanin purified from the cyanobacterial cells. ThepetE gene product is correctly processed in E. coli as deduced from the N-terminal amino acid sequences. These results, along with the identical physicochemical and kinetic properties of the two protein preparations, confirm that expression of petE in E. coli is an adequate tool to address the study of Synechocystis plastocyanin by site-directed mutagenesis.

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Section: Resultsmentioning

confidence: 99%

Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E. coli transformed cells are identical

Hervás¹,

Navarro²,

Navarro³

et al. 1993

FEBS Letters

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“…[26,40] Cyclic voltametry experiments were performed at varying pH and temperature as described in ref. [17].…”

Section: Methodsmentioning

confidence: 99%

Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

et al. 2005

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The L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(I) ion of the His87 ligand have been evaluated. The mutation-induced changes in transition enthalpy cannot be explained by electrostatic considerations. The existence of enthalpy/entropy (H/S) compensation within the protein series indicates that solvent-reorganization effects control the differences in transition thermodynamics. Once these compensating contributions are factorized out, the resulting modest differences in transition enthalpies turn out to be those that can be expected on purely electrostatic grounds. Therefore, this work shows that the acid transition in cupredoxins involves a reorganization of the H-bonding network within the hydration sphere of the molecule in the proximity of the metal center that dominates the observed transition thermodynamics and masks the differences that are due to protein-based effects.

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“…An explanation for the great variability, may be that the copper was lost during isolation. One way to reconstitute and stabilize copper proteins, such as plastocyanin and azurin during the isolation, is to add excess copper (Karlsson et al 1989, Nordling et al 1990). Therefore, to minimize the possible loss of copper during the isolation procedure, PS II particles were also isolated in the presence of 1 0~M CuSO 4.…”

Section: Resultsmentioning

confidence: 99%

The 28 kDa apoprotein of CP 26 in PS II binds copper

et al. 1993

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Photosystem II (PS II) particles isolated from spinach in the presence of 10 μM CuSO4 contained 1.2 copper/300 Chl that was resistant to EDTA. When CuSO4 was not added during the isolation, PS II particles contained variable amounts of copper resistant to EDTA (0.1-1.1 copper/300 Chl). No correlation was found between copper content and oxygen evolving capacity of the PS II particles. To identify the copper binding protein, we developed a fractionation procedure which included solubilisation of PS II particles followed by precipitation with polyethylene glycol. A 22-fold purification of copper with respect to protein was achieved for a 28 kDa protein. Partial amino acid sequence of a 13 kDa fragment, obtained after V8 (endo Glu-C) protease treatment, showed identity with CP 26 over a 14 amino acid stretch. EPR measurements on the purified protein suggest oxygen and/or nitrogen as ligands for copper but tend to exclude sulfur. We conclude that the 28 kDa apoprotein of CP 26 from spinach binds one copper per molecule of CP 26. A possible function for this copper protein in the xanthophyll cycle is discussed.

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Expression of spinach plastocyanin in E. coli

Cited by 30 publications

References 29 publications

Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E. coli transformed cells are identical

Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E. coli transformed cells are identical

Electrostatic Effects on the Thermodynamics of Protonation of Reduced Plastocyanin

The 28 kDa apoprotein of CP 26 in PS II binds copper

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