Anaerobic production and purification of recombinant algal hydrogenase

72 Pages·2016·17.4 MB·English

Anaerobic production and purification of
recombinant algal hydrogenase

Ole Boye

Master thesis in
Molecular Biosciences
(60 study points)

Department of Medical Biochemistry
Oslo University Hospital

UNIVERSITY OF OSLO
Faculty of Mathematics and Natural Sciences
Department of Biosciences

6/11/2016
I
Anaerobic production and purification of
recombinant algal hydrogenase

Ole Boye

II
Copyright Ole Boye

2016

Anaerobic production and purification of recombinant algal hydrogenase

Ole Boye

http://www.duo.uio.no

Trykk: Reprosentralen, Universitetet i Oslo

III
IV
Acknowledgements

This thesis concludes my Master's degree at the Institute of Biosciences, University of Oslo.
The research part of the study was carried out at the Department of Medical Biochemistry at
Rikshospitalet (Oslo University Hospital) from March 2015 to September 2016.

I would like to thank my supervisors, Bjørn Dalhus and Mingyi Yang, my internal supervisor
Hans-Petter Hersleth, and in particular my main supervisor Paul Hoff Backe, who was a
patient and supportive guide throughout the project. I would also like to thank Magnar Bjørås,
the leader of the research group at Oslo University Hospital. The two technicians at the lab,
Pernille Blicher and Lene Kittelsen, have been of great help.

The collaborators at UiO, led by Truls Norby, and at SINTEF, led by Sen Mei, have been
very helpful. Bente Tilset at SINTEF and Athanasios Eleftherios Chatzitakis at UiO, who
helped me with attachment experiments and gas chromatography, deserve my special thanks.

Oslo, November 2016

Ole Boye

V
Abbreviations

AP Alkaline phosphatase
BSA Bovine serum albumin
BT 2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol
CN Cyanide group
CO Carbonyl group
ECF Enhanced Chemifluorescence
EDTA Ethylenediaminetetraacetic acid
DLS Dynamic light scattering
F Phenylalanine
Fd Ferredoxin from Chlamydomonas reinhardtti
Fe Iron
GTP Guanine triphosphate
H Molecular hydrogen
2
HydA1 Hydrogenase from Chlamydomonas reinhardtti
HydE Maturation protein from Clostridium acetobutylicum
HydF Maturation protein from Clostridium acetobutylicum
HydG Maturation protein from Clostridium acetobutylicum
L Leucine
LB Lurio-Bertani medium
MOPS 3-(N-morpholino)propanesulfonic acid
MWNT Multi-walled carbon nanotubes
NaDT Sodium dithionite
NaOH Sodium hydroxide
Ni Nickel
O Molecular oxygen
2
PBS-T Phospho-buffered saline with 0.1% Tween-20
PCR Polymerase chain reaction
ROS Reactive oxygen species
rpm Rounds per minute
VI
S Sulfur
SDS Sodium dodecyl sulfate
SOC Super optimal broth with catabolite repression
TEV Tobacco etch virus
TGS Tris-Glycine SDS buffer
Tris Tris(hydroxymethyl)aminomethane
VII
Abstract

Hydrogen is an energy carrier that does not pollute when combusted, and is therefore ideal in
a green economy. However, current production of hydrogen is catalyzed by expensive and
noble metals, and a cheaper and non-polluting production method is desirable. Hydrogenase
is an enzyme that catalyzes the reversible formation of hydrogen by reduction of protons:

2H+ + 2e- = H
2

Hydrogenase can therefore potentially be used as a catalyst in the production of hydrogen,
replacing the noble metals currently used.

In this master thesis, I describe progress in producing hydrogen using a recombinant algal
hydrogenase as a catalyst. This work is part of a larger research project aiming to produce
hydrogen fuel in a solar cell by splitting water, using the hydrogenase as a bio-catalyst.
Several methods were used in the master project, including anaerobic protein expression in E.
coli, protein purification, mutagenesis and gas chromatography. Active hydrogenase enzyme
was successfully produced. The hydrogenase activity was verified by gas chromatography.
The enzyme was attached to several surfaces, in particular carbon nanotubes. In addition,
attempts were made to produce oxygen-resistant mutants. These advances fulfill several of
the subgoals of the project, in addition to bringing closer the realization of the combined solar
and hydrogen producing cell.

VIII
IX
Table of contents

1 Introduction .............................................................................................................................. 1
1.1 Hydrogen and fuel cells ............................................................................................................... 1
1.2 Production of hydrogen ............................................................................................................... 2
1.3 Research project: PH2BioCat ..................................................................................................... 3
1.4 Hydrogenases .................................................................................................................................. 5
1.5 Hydrogenases and oxygen .......................................................................................................... 7
1.6 HydA1 ................................................................................................................................................ 8
1.7 Aims ................................................................................................................................................. 12
2 Materials and methods ........................................................................................................ 13
2.1 Plasmid transformation in E. coli .......................................................................................... 13
2.2 Protein expression and solubility test ................................................................................. 14
2.2.1 Expression test ........................................................................................................................................ 15
2.2.2 Solubility test ........................................................................................................................................... 15
2.2.3 SDS-PAGE .................................................................................................................................................. 15
2.3 Expression of active protein ................................................................................................... 15
2.4 Protein purification ................................................................................................................... 16
2.4.1 Lysate preparation ................................................................................................................................ 16
2.4.2 Ion-exchange chromatography ........................................................................................................ 17
2.4.3 TEV cleavage ............................................................................................................................................ 17
2.4.4 Affinity chromatography .................................................................................................................... 17
2.5 Protein verification by Western blotting ............................................................................ 19
2.6 Protein yield ................................................................................................................................. 19
2.6.1 Protein concentration measurement ............................................................................................ 19
2.6.2 Concentration of protein .................................................................................................................... 20
2.7 Spectrophotometric hydrogenase activity assay ............................................................. 20
2.8 Gas chromatography hydrogenase activity assay ............................................................ 21
2.9 Protein size distribution, attachment and storage .......................................................... 22
2.9.1 Dynamic light scattering ..................................................................................................................... 22
2.9.2 Protein immobilization on carbon nanotubes .......................................................................... 22
2.9.3 Storage ........................................................................................................................................................ 23
2.10 Protein engineering ................................................................................................................. 23
2.10.1 Site-directed mutagenesis ............................................................................................................... 23
2.10.2 Miniprep for DNA plasmid extraction ........................................................................................ 25
2.10.3 Sequencing ............................................................................................................................................. 25
2.10.4 Tranformation of plasmids with mutated hydrogenase .................................................... 25
3 Results & discussion ............................................................................................................. 26
3.1 Plasmid transformation in E. coli .......................................................................................... 26
3.2 Protein expression and solubility test ................................................................................. 26
3.3 Expression of active protein ................................................................................................... 27
3.4 Protein purification ................................................................................................................... 29
3.4.1 Lysate isolation ....................................................................................................................................... 29
3.4.2 Ion-exchange chromatography ........................................................................................................ 29
3.4.3 TEV cleavage ............................................................................................................................................ 30
3.4.4 Affinity chromatography .................................................................................................................... 31
3.5 Protein verification by Western blotting ............................................................................ 33
X