Heterologous Production and Purification of a ... - ACS Publications

Subscriber access provided by READING UNIV

Letter

Heterologous production and purification of a functional chloroform reductive dehalogenase Bat-Erdene Jugder, Karl A.P. Payne, Karl Fisher, Susanne Bohl, Helene Lebhar, Mike Manefield, Matthew Lee, David Leys, and Christopher P. Marquis ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00846 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

1 2 3

Heterologous production and purification of a functional chloroform

4

reductive dehalogenase

5 6

Bat-Erdene Jugder†,‡, Karl A. P. Payne‡, Karl Fisher‡, Susanne Bohl†,§, Helene Lebhar†, Mike

7

Manefield¶,ς, Matthew Lee¶, David Leys‡ and Christopher P. Marquis†,*

8 9

†

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia, NSW 2052

10 ¶

School of Civil and Environmental Engineering, University of New South Wales, Sydney,

11

Australia, NSW 2052

12 ς

School of Chemical Engineering, University of New South Wales, Sydney, Australia,

13

NSW 2052

14 ‡

15 16

§

Manchester Institute of Biotechnology, University of Manchester, United Kingdom, M1 7 DN

Department of Biotechnology, Mannheim University of Applied Sciences, Mannheim, Germany

17 18

* Corresponding Author

19 20

ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

21

ABSTRACT

22

Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of

23

anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases

24

is desirable for structural and functional studies, however there are few reports of successful

25

expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred

26

electron acceptor is chloroform. This study describes efforts to express recombinant reductive

27

dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli

28

and Bacillus megaterium. Here we report the recombinant expression of soluble and functional

29

TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful

30

incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis

31

spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA

32

was demonstrated. This is the first known report of heterologous expression and purification of a

33

respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

34


Page 2 of 15

Page 3 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


35

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant, toxic and

36

carcinogenic environmental pollutants contaminating soil and groundwater. Organohalide

37

respiring bacteria (ORB) are a potential solution to remediate organohalide contaminated sites,

38

as they are capable of using these compounds as terminal electron acceptors for the generation of

39

cellular energy. Central to the process are reductive dehalogenases (RDase; EC 1.97.1.8), which

40

are membrane-associated enzymes that catalyze reductive dehalogenation reactions resulting in

41

the generation of lesser-halogenated compounds that may be less toxic and more biodegradable.

42

RDases are metalloenzymes, meaning their activity is dependent on two protein-associated

43

cofactors; cobamide and iron-sulfur clusters.1-3

44

Chloroform (CF), primarily used in the production of refrigerants, is a prevalent and

45

recalcitrant organohalide contaminant and its improper use and disposal has resulted in

46

groundwater contamination at numerous industrial sites globally.4 Its hazardous effect on the

47

environment, carcinogenicity and acute toxic effects on human health has prompted

48

bioremediation research towards its detoxification.5 Dehalobacter (Dhb) sp. strain UNSWDHB

49

is an obligate ORB capable of respiring CF, thereby transforming it to dichloromethane (DCM).6

50

Strain UNSWDHB

51

respiration.7 Native TmrA was produced and purified from the membrane fraction of

52

UNSWDHB cells to apparent homogeneity, using detergent-based membrane solubilisation and

53

anion exchange chromatographic purification.8 This allowed further biochemical characterization

54

of the purified enzyme. However, ORB are slow growing with low cell yields. This coupled with

55

non-trivial culture maintenance make routine RDase production and purification from the native

56

source difficult.

produces a CF-RDase, (TmrA), which is highly expressed during CF



Page 4 of 15

57

The heterologous expression of respiratory RDases has been reported to be extremely

58

challenging. Several efforts to heterologously express functional RDases have failed over the

59

past 20 years, mainly due to the absence of one or both cofactors (a corrinoid and two Fe-S

60

clusters) in the recombinant proteins.2, 3 The co-expression of solubility enhancing tags, such as

61

trigger factor (TF), was reported to drive soluble expression of PceA fusion proteins from

62

Dehalobacter restrictus in Escherichia coli, however, no enzymatic activity was observed.9 The

63

first demonstration of functional expression of a recombinant RDase was reported for PceA from

64

Desulfitobacterium hafniense Y51 produced in Shimwellia blattae, a bacterium capable of de

65

novo synthesis of cobalamin.10 Co-expression of PceT, a chaperone protein, enhanced the

66

overexpression of functional PceA, as demonstrated in the E. coli crude extracts. However, an

67

attempt to purify the recombinant PceA failed, as the expressed protein did not bind to the Strep-

68

Tag column.

69

In this study, heterologous expression and purification of TmrA in a soluble and

70

functional form was attempted. Initially, molecular cloning and expression studies of the enzyme

71

in E. coli were undertaken. For this, the TAT signal and predicted membrane spanning regions

72

were removed from the tmrA gene prior to gene synthesis and cloning; a strategy to direct

73

expression of otherwise periplasmic RDases towards the cytoplasm (Figure 1A).9,

74

rhamnose-inducible heterologous expression of TmrA under the control of a tunable rhaBAD

75

promoter was investigated (Figure 1B). Whilst slightly tunable protein expression with

76

increasing concentrations of rhamnose was observed in pD864-TmrA transformed cells, protein

77

expression in general was not tightly regulated and TmrA was expressed in an insoluble form

78

(Figure S1).

79


11

First,

Page 5 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 81

82 83

Figure 1. Schematic of the heterologous expression of TmrA. (A) The region framed in red dashed line

84

containing TAT signal peptide and a predicted transmembrane domain was removed prior to tmrA gene

85

cloning. (B) TmrA expression in E. coli under rhamnose- and IPTG-inducible promoters in pD864 and

86

pOPIN vectors, respectively. TmrA fusion constructs with solubility (SUMO, TRX, NusA, MBP and TF)

87

and affinity (NHis and CHis) tags encoded in pOPIN expression vectors were constructed and

88

investigated. 3C is the recognition sequence for the Human Rhinovirus 3C Protease to allow the cleavage

89

of the fusion proteins. Cofactor reconstitution and refolding were attempted on pOPIN-CHis-TmrA

90

construct. (C) A functional TmrA was expressed in B. megaterium containing pT7-RNAP plasmid that

91

permits xylose-inducible expression of T7 polymerase. (D) Production and purification of TmrA in B.

92

megaterium



93

Next our attention turned to the heterologous expression of TmrA under the control of an

94

IPTG-inducible system using the pOPIN vector system with a T7 inducible promoter and several

95

fusion tags (Figure 1B). The co-expression of hydrophilic fusion partners has been shown to be

96

an effective tool to increase the amount of soluble recombinant protein. Numerous expression

97

screens were carried out and it was shown that TmrA in fusion with affinity (N- or C-terminal

98

His) and solubility tags (SUMO, Trx, NusA, MBP and TF) can be overexpressed at 37°C from

99

all constructs (Figure S2). However, none of our experiments yielded soluble recombinant

100

protein with any of the constructs, despite attempting induction at a lower temperature, varying

101

the growth media and IPTG concentration nor use of cofactor supplementation (Figure S3 and

102

S4). Identical solubility tags to those used in the present study (SUMO, NusA, MBP and TF) had

103

been shown previously to overexpress PceA from D. restrictus in E. coli9. However, the

104

recombinant PceA was catalytically inactive, possibly due to insufficient or incorrect cofactor

105

incorporation. Heterologous expression of RDases fused to solubility tags leading to insoluble

106

and non-functional enzymes has also been reported elsewhere. For example, PceA from D.

107

hafniense strain Y51 was investigated and overexpressed in fusion with Trx, S-tag for antibody

108

recognition and N- and C-terminal His tags in E. coli.12 The fusion protein was also expressed in

109

an insoluble form and denaturation followed by refolding of the fusion protein did not recover

110

dechlorination activity.

111

To date, no respiratory RDase in a soluble and catalytically active form has been

112

heterologously expressed in E. coli. In this study, we also were not able to obtain a soluble TmrA

113

in E. coli. Recently, a vinyl chloride-reducing dehalogenase VcrA from Dehalococcoides

114

mccartyi strain VS was expressed in E. coli in an insoluble form but recovered from inclusion

115

bodies via denaturing purification followed by chemical reconstitution of cofactors during


Page 6 of 15

Page 7 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


116

protein refolding to an active form.11 Since no soluble TmrA was expressed throughout extensive

117

expression studies, it was decided to investigate purification of detergent-denatured TmrA

118

protein from the cellular insoluble fraction with subsequent refolding. For this, the

119

pOPIN-TmrA-CHis transformant was employed due to its higher expression levels in 2YT media

120

(compared to LB media) at 37°C. Five liter fermentations were undertaken with or without

121

external cofactor supplementation. Adenosylcobalamin was used as a corrinoid cofactor based on

122

previous reports of attempts to produce recombinant RDases.10, 11 Heterologously expressed C-

123

His-tagged TmrA was captured by Ni-NTA affinity chromatography under denaturing conditions

124

(8 mM urea) and subjected to several refolding methods, such as refolding by flash or slow

125

dilution, by dialysis, on-column refolding and chemical cofactor reconstitution. Regardless of

126

efforts undertaken in this work, using a similar approach to others for protein refolding11, no

127

refolded and active TmrA could be obtained, mainly due to precipitation issues and subsequent

128

protein loss.

129

Our focus then shifted to heterologously expressing TmrA in Bacillus megaterium, a

130

bacterium known to possess de novo cobalamin biosynthesis capacity. Recently, a co-metabolic

131

(non-respiratory) reductive dehalogenase, NpRdhA, from Nitratireductor pacificus strain pht-3B

132

was heterologously expressed in B. megaterium.13 The C-terminal His-tagged NpRdhA was

133

obtained in an active form in aerobically grown B. megaterium under a xylose-inducible

134

promoter. Although the recombinant NpRdhA represents a phylogenetically distinct and aerobic

135

RdhA, as opposed to respiratory RDases, it allowed further studies to reveal RDase structure and

136

generation of a proposed enzymatic mechanism. In this study, we utilized the same molecular

137

cloning and expression tools for B. megaterium to express the respiratory TmrA.



138

The tmrA gene with BsrGI and BamHI sites and N-terminal His-tag were PCR-amplified

139

and cloned into the same restriction sites of pPT7 plasmid using the In-Fusion cloning technique

140

(Clontech). The pPT7 plasmid harboring the tmrA gene (pPT7-tmrA, Figure 1C) was then was

141

transformed into B. megaterium MS941 protoplasts containing the pT7-RNAP plasmid that

142

permits xylose-inducible expression of T7 polymerase. Xylose-inducible expression of TmrA

143

was performed aerobically in TB media at 17°C. The cells were disrupted anaerobically using a

144

French press (Figure 1D). Following anaerobic ultracentrifugation of cell lysate, protein SDS-

145

PAGE and western blotting analyses revealed TmrA expressed in a soluble form (Figure 2A and

146

B), which was further confirmed by tryptic digest and liquid chromatography tandem mass

147

spectrometry (LC-MS/MS) at the UNSW Biomedical Mass Spectrometry Facility.

148


Page 8 of 15

Page 9 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


149

150 151

Figure 2. Heterologously expressed TmrA. (A) SDS-PAGE of purification samples, including soluble

152

fraction, IMAC (Ni-NTA) and ResQ column eluted proteins and Western Blot of the purified protein

153

(ResQ column) with His6 Tag Monoclonal Antibody, HRP conjugate (Invitrogen). (B) UV-visible

154

absorption spectra of the purified TmrA and the cobalamin extracted from the enzyme (inset). (C) Gas

155

chromatogram of CF (RT=5.044 min) dechlorination reaction by the purified TmrA to produce DCM (in

156

blue; RT=4.159 min) overlapped with a heat-deactivated negative control (in grey). The data represents

157

the dechlorination activity after incubating the enzymatic reaction at 30°C for 5 h, during which in total of

158

823 nmol DCM was produced. The peak heights (pA) for CF, DCM from the TmrA-catalyzed reaction

159

and CF from the negative control are given as CF pATmrA, DCM pATmrA and CF pANC, respectively.

160

To purify NHis-tagged TmrA, the soluble fraction resulting from a 15-L fermenter was

161

loaded onto a Ni-NTA drip column under anaerobic conditions. The TmrA protein was eluted

162

with approximately 50 mM Imidazole-containing buffer and verified by SDS-PAGE and western

163

blotting (Figure 2A and B). To improve the purity of the protein preparation, an anion exchange

164

ResQ column was used. For this, the Ni-NTA eluate was buffer-exchanged and loaded into the

165

ResQ column aerobically using an ÄKTAexplorer. At approximately 250 mM NaCl, the TmrA

166

protein was recovered from a single peak (Figure 2A and B).

167

168

Next, UV-visible spectroscopy, was used to detect the cofactors incorporated into

169

purified RDases. The heterologously expressed TmrA exhibited a broad absorbance between 400

170

and 500 nm with the absorption maximum at 420 nm being indicative for the [4Fe-4S] clusters

171

(Figure 2C), which is a common feature for all previously reported RDases,14-16 including the

172

native version of TmrA.8 Cyanide extraction of TmrA-associated cobalamin revealed the

173

absorption maxima at 361, 522 and 550 nm which is typical for cyanocobalamin (inset in Figure



Page 10 of 15

174

2C).14-16 A stoichiometry of 0.52 ± 0.03 mol of corrinoid per mol of TmrA was determined in the

175

pink solution extracted by means of cyanolysis. The Fe content of the dehalogenase using a

176

bathophenanthroline assay revealed 7.26 ± 0.48 mol of Fe/mol of protein, which is close to the

177

expected value of 8.

178

To confirm the catalytic activity of TmrA, a CF reductive dehalogenating activity assay

179

was performed using Ti(III) citrate as the bulk electron donor and methyl viologen as an electron

180

transfer mediator.8 Activity was monitored by the production of DCM. CF dechlorination

181

activity was found in the soluble protein fraction (0.1 U mg of protein–1) and the Ni-NTA eluted

182

protein (30.2 U mg of protein–1). The final aerobic ResQ-purified protein exhibited CF reductive

183

dehalogenation activity at a specific activity of 110 U mg of protein–1 (Figure 2D and Table 1).

184

This activity is 11-fold lower than the activity of native TmrA previously determined

185

(1.27 × 103 U mg of protein–1).8 This might be explained by a number of factors, including the

186

presence of apoenzymes that have not correctly incorporated corrinoid cofactors, the failure to

187

co-express a trigger factor to facilitate correct folding or partial oxidation of enzymes during

188

purification. Our data suggests that the reduced specific activity of the recombinant enzyme

189

(compared to purified enzyme from UNSWDHB), can only be in part attributed incomplete

190

incorporation of corrinoid, since the observed cobalamin:enzyme molar ratio is approximately

191

half the expected value of 1. Heat-deactivated (incubating extracts at 80°C for 10 min)

192

B. megaterium crude protein extracts and enzyme-free reactions were assayed as negative

193

controls to rule out non-enzymatic CF reductive activity and no dechlorination activity was

194

observed in such negative controls (Figure 2C).

Soluble fraction

Total activity (nmoles/min)

Yield (%)

Total protein (mg)

Specific activity (nmoles/min/mg)

Purification factor

278.6

100

2700.0

0.1

1


Page 11 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ni-NTA eluate Res-Q eluate

158.5 99.0

57 36

5.25 0.9

30.2 110.0

293 1066

195

196 197

Table 1

198

In conclusion, an extensive study to heterologously express TmrA was conducted under

199

several expression conditions. Firstly, a TmrA expression study in E. coli was undertaken by

200

employing several strategies to enhance soluble expression, such as removal of a TAT signal

201

peptide, rhamnose or IPTG-controlled induction, co-expression with commonly used solubility-

202

enhancing protein tags and expression screens under numerous conditions. Despite all the efforts

203

to obtain a soluble TmrA, as well as cofactor reconstitution attempts on the denatured protein,

204

E. coli expression was unsuccessful. However, using the xylose-inducible expression system in

205

the corrinoid-producing B. megaterium as an expression host, soluble and functional TmrA was

206

successfully expressed and purified in active form via two liquid chromatographic steps. The

207

presence of both cofactors was revealed by the UV-vis spectra recorded for the purified enzyme

208

and metal analysis undertaken. The purified protein exhibited CF dechlorination activity in vitro.

209

As a result, a membrane-associated respiratory RDase has been heterologously expressed in a

210

soluble and active form and purified for the first time.

211

METHODS

212

The detailed experimental procedures of tmrA gene cloning for E. coli and B. megaterium

213

heterologous protein expression, solubility screenings of TmrA expressed in E. coli, production,

214

denaturing purification and refolding of the TmrA-CHis variant, heterologous expression of

215

TmrA in B. megaterium and its purification, protein concentration determination, SDS-PAGE

Activity, yield and purification factor of the soluble extract, Ni-NTA eluate and anion exchange fractions



216

and Western blotting, UV-Vis measurement, metal analysis in the recombinant enzyme and gas

217

chromatographic activity assay are provided in the Supporting Information.

218 219

ASSOCIATED CONTENT

220

Supporting Information Available: Detailed experimental procedures, including gene cloning,

221

protein expression and purification, protein refolding and protein characterization are available

222

as Supplementary Information. This material is available free of charge via the internet at

223

http://pubs.acs.org.

224 225

AUTHOR INFORMATION

226

Corresponding Author

227

*Phone: +612 93853898, E-mail: [email protected]

228

Notes

229

The authors declare no competing financial interest.

230 231

ACKNOWLEDGEMENTS

232

We acknowledge the PEF at the University of Queensland, Australia for cloning the tmrA gene

233

into pOPIN vectors with solubility tags. We also acknowledge the University of New South

234

Wales (UNSW) for scholarship and travel support for BJ. DL is a Royal Society Wolfson

235

Research Merit Award holder.

236 237

REFERENCES

238 239

1. Leys, D., Adrian, L., and Smidt, H. (2013) Organohalide respiration: microbes breathing chlorinated molecules, Philos. Trans. Soc., B 368, 20120316.


Page 12 of 15

Page 13 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277


2. Jugder, B. E., Ertan, H., Lee, M., Manefield, M., and Marquis, C. P. (2015) Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides, Trends Biotechnol. 33, 595-610. 3. Jugder, B. E., Ertan, H., Bohl, S., Lee, M., Marquis, C. P., and Manefield, M. (2016) Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation, Front. Microbiol. 7, 249. 4. Koenig, J., Lee, M., and Manefield, M. (2015) Aliphatic organochlorine degradation in subsurface environments, Rev.Environ. Sci. Bio/Technol. 14, 49-71. 5. ASTDR. (2015) Toxological profile for chloroform. http://www.atsdr.cdc.gov/ToxProfiles/tp6-c4.pdf 2015., (accessed 1 April, 2017). 6. Wong, Y. K., Holland, S. I., Ertan, H., Manefield, M., and Lee, M. (2016) Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration, Environ. Microbiol. 18, 3092-3105. 7. Jugder, B. E., Ertan, H., Wong, Y. K., Braidy, N., Manefield, M., Marquis, C. P., and Lee, M. (2016) Genomic, transcriptomic and proteomic analyses of Dehalobacter UNSWDHB in response to chloroform, Environ. Microbiol. Reports 8(5), 814-824. 8. Jugder, B. E., Bohl, S., Lebhar, H., Healey, R. D., Manefield, M., Marquis, C. P., and Lee, M. (2017) A bacterial chloroform reductive dehalogenase: purification and biochemical characterization, Microb. Biotechnol. 10(6),1640-1648 9. Sjuts, H., Fisher, K., Dunstan, M. S., Rigby, S. E., and Leys, D. (2012) Heterologous expression, purification and cofactor reconstitution of the reductive dehalogenase PceA from Dehalobacter restrictus, Protein Expression Purif. 85, 224-229. 10. Mac Nelly, A., Kai, M., Svatos, A., Diekert, G., and Schubert, T. (2014) Functional heterologous production of reductive dehalogenases from Desulfitobacterium hafniense strains, Appl.Environ. Microbiol. 80, 4313-4322. 11. Parthasarathy, A., Stich, T. A., Lohner, S. T., Lesnefsky, A., Britt, R. D., and Spormann, A. M. (2015) Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS, J. Am. Chem. Soc. 137, 3525-3532. 12. Suyama, A., Yamashita, M., Yoshino, S., and Furukawa, K. (2002) Molecular characterization of the PceA reductive dehalogenase of Desulfitobacterium sp. strain Y51, J.Bacteriol. 184, 3419-3425. 13. Payne, K. A., Quezada, C. P., Fisher, K., Dunstan, M. S., Collins, F. A., Sjuts, H., Levy, C., Hay, S., Rigby, S. E., and Leys, D. (2015) Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation, Nature 517, 513-516. 14. Neumann, A., Wohlfarth, G., and Diekert, G. (1996) Purification and characterization of tetrachloroethene reductive dehalogenase from Dehalospirillum multivorans, J. Biol.Chem. 271, 16515-16519.



278 279 280 281 282 283 284

15. Schumacher, W., Holliger, C., Zehnder, A. J., and Hagen, W. R. (1997) Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachloroethene reductase of Dehalobacter restrictus, FEBS Lett. 409, 421-425. 16. Christiansen, N., Ahring, B. K., Wohlfarth, G., and Diekert, G. (1998) Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense, FEBS Lett. 436, 159-162.


Page 14 of 15

Page 15 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Soluble and active expression of a recombinant reductive dehalogenase 342x195mm (150 x 150 DPI)


Heterologous Production and Purification of a ... - ACS Publications

Recommend Documents