Expression of Cowpea Chlorotic Mottle Virus Capsids in Pichia pastoris and their Application in Nanotechnology
- Issued Date
- 2013
- Abstract
- Viruses and noninfectious virus-like particles (VLPs) exhibit the characteristics of ideal building blocks with highly symmetrical, polyvalent, and monodisperse structures for their utilization in nanotechnology. Cowpea chlorotic mottle virus (CCMV) is icosahedral virus with a diameter of 28 nm which has been a model system for virus studies for over 40 years. Recently CCMV has been considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self-assemble into VLPs in vitro makes it suitable reaction vessel for nanomaterial encapsulation and modification.
Chapter-1 of this thesis is the general introduction of virus and virus capsids, and their applications in nanotechnology.
Chapter-2 is the backgrounds and objectives concerning the originality of the thesis.
In Chapter-3, the coat protein (CP) gene of CCMV was firstly optimized according to codon perference and expressed in Pichia pastoris GS115. The synthesized CP gene (573 bp) was cloned into Pichia shuttle vector pPICZ A under the alcohol oxidase I (AOX1) promoter. The recombinant plasmid pPICZ A-CPsyns was transformed into P. pastoris GS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. Transformant GS115-27-6 was selected for scale-up fermentation in 5-L fermenter and CCMV CP yields reached up to 4.8 mg/mL. The CCMV VLPs were purified by modified poly(ethylene glycol) (PEG) precipitation followed by cesium chloride density gradient ultracentrifugation. The assembled CCMV VLPs were analyzed by UV spectrometry and transmission electron microscopy (TEM). The resulting data indicated the production of CCMV capsids by P. pastoris fermentation available for utilization in pharmacology or nanotechnology fields.
In Chapter-4, CCMV capsids were employed to encapsulate Prussian blue (PB, MFeIII[FeII(CN)6] (M = NH4, Li, Na, K)) particles based on electrostatic interaction. A negatively charged metal complex, hexacyanoferrate (III), was entrapped inside the capsids through the disassembly/reassembly process under pH change from 7.5 to 5.2. The loaded capsids acted as nanoreactors because the metal complex could react with a second metal ion, iron (II), to fabricate PB particles in protein cages. The synthesis of PB in CCMV capsids was confirmed by unique colour transition of Prussian blue, UV-vis spectrum at 710 nm, and size exclusion fast protein liquid chromatography (FPLC). TEM image PB-CCMV biohybrids presented discrete spherical particles with relatively homogeneous size. The hydrodynamic diameters of PB-CCMV were measured by dynamic light scattering (DLS) showing two peaks of 29.2±1.7 nm corresponding to T = 3 particles, and 17.5±1.2 nm corresponding to pseudo T = 2 particles. The encapsulation and synthesis of PB in CCMV provided a fast and efficient method for the self-organization of homo- and heterobimetallic nanoparticles.
In Chapter-5, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was exploited to the exterior surface of CCMV for the first time. The exposed carboxyl residues of CCMV were addressed with alkyne and further modified with azide through triazole connection in the presence of CuSO4, TCEP, and BCDS. Fluorogenic coumarin was successfully grafted on CCMV and monitored by FPLC and UV irradiated SDS-PAGE. Oligo-ethylene glycol (OEG) short chain and RGD (Arg-Gly-Asp) peptide were also installed on CCMV via CuAAC reaction. FPLC, TEM, and DLS analysis verified the modification and integrity of viral capsids. Interestingly OEG-CCMV displayed a distinct phenomenon of connected bridges with the intact capsids cross-linked with each other. Wild type CCMV, OEG-CCMV, and RGD-CCMV were absorbed onto APTES slides for the cell binding with HeLa cells. The opposite adhesion behaviors between OEG-CCMV and RGD-CCMV indicated the inhibition effect of OEG and promotion effect of RGD on cell attachment. As the most widely recognized example of click chemistry, CuAAC reaction paved a brilliant way for the design of bionanoparticle-based nanosensor, drug delivery carrier, and tissue engineering materials.
In the appendix I, Heterocapsa circularisquama RNA virus (HcRNAV) 109 CP gene was expressed in P. pastoris using the same strategy as CCMV. The encapsulation of fluorescence dye-labeled myoglobin by self-assembled HcRNAV capsids demonstrated potential application in the harmful algae blooms (HABs) control. The co-expression of HcRNAV CP and algicidal peptide PMAP-23-D7 was also accomplished with a structure transition from polyhedral to rod-like particles which might stem from the insertion of PMAP-23-D7 at N-terminus of HcRNAV CP.
In the appendix II, an efficient method for Pichia cell disruption that employed aminopropyl magnesium phyllosilicate (AMP) clay-assisted glass beads mill was presented. AMP clay is functionalized nanocomposite resembling the talc parent structure Si8Mg6O20(OH)4. The total protein concentration reached 4.24 mg/mL after 4 minutes treatment by glass beads mill combined with 0.2 % AMP clay, which was 11.2 % higher compared to glass beads mill only and the time was half shortened. The stability of purified CCMV VLPs illustrated it could be a reliable method for the disruption of yeast cell.
In conclusion, the expression of virus capsids in P. pastoris was established as a simple and efficient approach. The encapsulation and modification of the capsids were achieved for their applications in pharmaceutics and nanotechnology.
- Alternative Title
- Expression of Cowpea Chlorotic Mottle Virus Capsids in Pichia pastoris and their Application in Nanotechnology
- Alternative Author(s)
- Yuanzheng Wu
- Affiliation
- 조선대학교 대학원
- Department
- 일반대학원 화학공학과
- Advisor
- 신현재
- Awarded Date
- 2013-08
- Table Of Contents
- List of Tables.............................................................................viii
List of Figures..............................................................................ix
Abstract in English....................................................................xiv
Chapter 1. Introduction...............................................................1
1.1 Virus and virus capsids...........................................................................1
1.1.1 CCMV................................................................................................................5
1.1.2 CPMV................................................................................................................9
1.1.3 TMV...................................................................................................................9
1.2 Surface modification of virus capsids..................................................10
1.2.1 NHS-ester acylation.........................................................................................12
1.2.2 CuAAC or click chemistry...............................................................................12
1.2.3 Hydrazone ligation...........................................................................................13
1.3 Encapsulation by virus capsids.............................................................14
1.3.1 Electrostatic interaction....................................................................................17
1.3.2 Chemical conjugation.......................................................................................18
1.3.3 Covalent attachment by genetic manipulation.................................................19
1.4 Application of VLPs in nanotechnology..............................................20
1.4.1 Drug delivery...................................................................................................20
1.4.2 Single-enzyme nanoreactor..............................................................................23
1.4.3 In vivo imaging.................................................................................................23
1.4.4 Light-harvesting cells.......................................................................................24
1.5 Future directions....................................................................................25
Chapter 2. Originality of Thesis...............................................27
2.1 Backgrounds...........................................................................................27
2.2 Objectives...............................................................................................32
Chapter 3. Expression and Self-assembly of CCMV in Pichia pastoris.........................................................................................35
3.1 Introduction............................................................................................35
3.2 Materials and methods..........................................................................41
3.2.1 Synthesis of P. pastoris codon-optimized CCMV CP.....................................41
3.2.2 Construction of recombinant plasmid..............................................................41
3.2.3 Expression of CCMV CP in P. pastoris...........................................................41
3.2.4 Scale-up fermentation......................................................................................42
3.2.5 Purification of CCMV VLPs............................................................................43
3.2.6 Analysis of purified CCMV VLPs...................................................................43
3.3 Results and discussion...........................................................................44
3.3.1 Synthesis of P. pastoris codon preferred CCMV CP.......................................44
3.3.2 Construction of recombinant plasmid..............................................................46
3.3.3 Expression of CCMV CP in P. pastoris...........................................................49
3.3.4 Scale-up fermentation......................................................................................51
3.3.5 Purification of CCMV VLPs............................................................................54
3.3.6 Analysis of purified CCMV VLPs...................................................................56
3.4 Summary.................................................................................................59
Chapter 4. Encapsulation of Prussian Blue by CCMV..........60
4.1 Introduction............................................................................................60
4.2 Materials and methods..........................................................................65
4.2.1 Preparation of disassembled CCMV solution..................................................65
4.2.2 Encapsulation and synthesis of Prussian blue nanoparticles by CCMV..........65
4.2.3 Purification of PB-CCMV by FPLC................................................................66
4.2.4 Analysis of the purified PB-CCMV.................................................................66
4.3 Results and discussion...........................................................................66
4.3.1 Encapsulation and synthesis of Prussian blue nanoparticles by CCMV..........66
4.3.2 Purification of PB-CCMV by FPLC................................................................70
4.3.3 Analysis of the purified PB-CCMV.................................................................72
4.4 Summary.................................................................................................75
Chapter 5. Surface Modification of CCMV by CuAAC Reaction.......................................................................................76
5.1 Introduction............................................................................................76
5.2 Materials and methods..........................................................................81
5.2.1 Materials and instruments................................................................................81
5.2.2 CCMV bioconjugation protocol.......................................................................81
5.2.3 Immobilization of modified CCMV on APTES slides....................................82
5.2.4 Cell adhesion....................................................................................................82
5.3 Results and discussion...........................................................................83
5.3.1 Introduction of alkyne group on CCMV..........................................................83
5.3.2 CuAAC reaction on CCMV-alkyne capsid......................................................84
5.3.3 Immobilization of on APTES slides................................................................93
5.3.4 Cell adhesion....................................................................................................95
5.4 Summary.................................................................................................98
Appendix I. Expression of HcRNAV and Co-expression with PMAP-23-D7 in P. pastoris........................................................99
6.1 Introduction............................................................................................99
6.2 Materials and methods........................................................................104
6.2.1 Synthesis of P. pastoris codon-optimized HcRNAV CPHsyns.....................104
6.2.2 Insertion of peptide PMAP-23-D7 at N-terminus of HcRNAV109 CPHsys.104
6.2.3 Construction of the recombinant vector pPICZ A-CPHsyns.........................106
6.2.4 Transformation of P. pastoris and cultivation of HCRNAV CPHsyns.........106
6.2.5 Purification of HcRNAV and HcRNAV-PMAP VLPs..................................106
6.2.6 Analysis of purified HcRNAV and HcRNAV-PMAP VLPs.........................107
6.2.7 Encapsulation of fluorescence dye-labeled myoglobin..................................107
6.3 Results and discussion.........................................................................107
6.3.1 Construction of expression vector pPICZ A-CPHsyns..................................107
6.3.2 Insertion of peptide PMAP-23-D7 at N-terminus of HcRNAV109 CPHsys.111
6.3.3 Expression of the HcRNAV CPHsyns gene in P. pastoris............................113
6.3.4 Purification of HcRNAV and HcRNAV-PMAP VLPs..................................115
6.3.5 Characterization of purified HcRNAV and HcRNAV-PMAP VLPs............117
6.3.6 Encapsulation of fluorescence dye-labeled myoglobin..................................120
6.4 Summary...............................................................................................123
Appendix II. Improved Cell Disruption of P. pastoris Utilizing Aminopropyl Magnesium Phyllosilicate (AMP) Clay...........124
7.1 Introduction..........................................................................................124
7.2 Materials and methods........................................................................126
7.2.1 Culture of P. pastoris transformant of CCMV CP.........................................126
7.2.2 Disruption of P. pastoris cells........................................................................126
7.2.3 Analysis of disrupted P. pastoris cells............................................................126
7.2.4 Evaluation of effect on CCMV CP by added AMP-clay................................127
7.3 Results and discussion.........................................................................127
7.3.1 Disruption of P. pastoris cells........................................................................127
7.3.2 Analysis of disrupted P. pastoris cells............................................................129
7.3.3 Evaluation of effect on CCMV CP by added AMP-clay................................132
7.4 Summary...............................................................................................135
Overall Conclusions and Future Aspects...............................136
References.................................................................................137
Abstract in Korean...................................................................158
List of Publications...................................................................163
Scientific Activities...................................................................164
Curriculum Vitae.....................................................................165
Acknowledgements...................................................................166
- Degree
- Doctor
- Publisher
- 조선대학교 대학원
- Citation
- 오원정. (2013). Expression of Cowpea Chlorotic Mottle Virus Capsids in Pichia pastoris and their Application in Nanotechnology.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/9861
http://chosun.dcollection.net/common/orgView/200000263907
- Appears in Collections:
- General Graduate School > 4. Theses(Ph.D)
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