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High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, Cramer SP, Swartz JR - PLoS ONE (2010)

Bottom Line: Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation.Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified.These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, USA.

ABSTRACT

Background: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.

Principal findings: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms.

Significance: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

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Fourier transform infrared spectra of heterologous [FeFe] hydrogenases produced in E. coli.Infrared spectra are for 100–200 µM of the (A) HydA1 and (B) CpI hydrogenases. Both enzymes were examined [1] in their as-isolated state as well as [2] following treatment with exogenous CO. Vibrational energies (in cm−1) for the H-cluster CO and CN− ligands are indicated in each spectrum. The wavenumber ranges for terminal CN− (ν(CN−)), terminal CO (ν(CO)), and bridging CO (ν(µ-CO)) vibrational modes are shown above the spectra. Scale bars shown at 1870 cm−1 represent a difference of 0.5 milliabsorbance units.
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pone-0015491-g003: Fourier transform infrared spectra of heterologous [FeFe] hydrogenases produced in E. coli.Infrared spectra are for 100–200 µM of the (A) HydA1 and (B) CpI hydrogenases. Both enzymes were examined [1] in their as-isolated state as well as [2] following treatment with exogenous CO. Vibrational energies (in cm−1) for the H-cluster CO and CN− ligands are indicated in each spectrum. The wavenumber ranges for terminal CN− (ν(CN−)), terminal CO (ν(CO)), and bridging CO (ν(µ-CO)) vibrational modes are shown above the spectra. Scale bars shown at 1870 cm−1 represent a difference of 0.5 milliabsorbance units.

Mentions: FTIR spectroscopy was used to analyze the purified HydA1 (Fig. 3A) and CpI hydrogenases (Fig. 3B) in both the as-isolated state as well as following treatment with exogenous CO. The spectra for both as-isolated hydrogenases clearly show peaks representing CN− and CO vibrational stretches, indicating the presence of fully assembled H-clusters. Based on previous reports for each of these enzymes [31], [32] as well as other [FeFe] hydrogenases [33], these spectra also indicate that the as-isolated hydrogenases have a mixture of H-clusters in both the oxidized (Hox) and reduced (Hred) states. The presence of DTH in the elution buffer was essential to prevent hydrogenase inactivation during purification, and this additive was likely the cause for the mixture of H-cluster redox states. The CO inhibition studies confirmed the presence of the H-cluster cofactors, as the CO and CN− vibrational modes shifted as expected after exogenous CO binding to the H-cluster.


High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, Cramer SP, Swartz JR - PLoS ONE (2010)

Fourier transform infrared spectra of heterologous [FeFe] hydrogenases produced in E. coli.Infrared spectra are for 100–200 µM of the (A) HydA1 and (B) CpI hydrogenases. Both enzymes were examined [1] in their as-isolated state as well as [2] following treatment with exogenous CO. Vibrational energies (in cm−1) for the H-cluster CO and CN− ligands are indicated in each spectrum. The wavenumber ranges for terminal CN− (ν(CN−)), terminal CO (ν(CO)), and bridging CO (ν(µ-CO)) vibrational modes are shown above the spectra. Scale bars shown at 1870 cm−1 represent a difference of 0.5 milliabsorbance units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991362&req=5

pone-0015491-g003: Fourier transform infrared spectra of heterologous [FeFe] hydrogenases produced in E. coli.Infrared spectra are for 100–200 µM of the (A) HydA1 and (B) CpI hydrogenases. Both enzymes were examined [1] in their as-isolated state as well as [2] following treatment with exogenous CO. Vibrational energies (in cm−1) for the H-cluster CO and CN− ligands are indicated in each spectrum. The wavenumber ranges for terminal CN− (ν(CN−)), terminal CO (ν(CO)), and bridging CO (ν(µ-CO)) vibrational modes are shown above the spectra. Scale bars shown at 1870 cm−1 represent a difference of 0.5 milliabsorbance units.
Mentions: FTIR spectroscopy was used to analyze the purified HydA1 (Fig. 3A) and CpI hydrogenases (Fig. 3B) in both the as-isolated state as well as following treatment with exogenous CO. The spectra for both as-isolated hydrogenases clearly show peaks representing CN− and CO vibrational stretches, indicating the presence of fully assembled H-clusters. Based on previous reports for each of these enzymes [31], [32] as well as other [FeFe] hydrogenases [33], these spectra also indicate that the as-isolated hydrogenases have a mixture of H-clusters in both the oxidized (Hox) and reduced (Hred) states. The presence of DTH in the elution buffer was essential to prevent hydrogenase inactivation during purification, and this additive was likely the cause for the mixture of H-cluster redox states. The CO inhibition studies confirmed the presence of the H-cluster cofactors, as the CO and CN− vibrational modes shifted as expected after exogenous CO binding to the H-cluster.

Bottom Line: Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation.Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified.These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, USA.

ABSTRACT

Background: The realization of hydrogenase-based technologies for renewable H(2) production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases.

Principal findings: In this report, we describe an improved Escherichia coli-based expression system capable of producing 8-30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H(2) evolution with rates comparable to those of enzymes isolated from their respective native organisms.

Significance: The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H(2)-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

Show MeSH
Related in: MedlinePlus