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Hamburg University of Applied Sciences
Faculty of Life Sciences
Master of Science in Pharmaceutical Biotechnology
Master Thesis
Optimization of Virus Amplification and Protein
Expression using Recombinant Baculoviruses
Date of Submission:
07.10.2019
Submitted by Urvish Desai, Enrollment No.:
First examiner: Prof. Dr. phil. nat. Oliver Ullrich
Second examiner: Prof. Dr. rer. nat. Birger Anspach
This thesis was performed and supervised in the laboratory of molecular biology
and cell culture techniques at Hamburg University of Applied Sciences.
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Acknowledgements
I would like to thank Prof. Dr. phil. nat. Oliver Ullrich, who provided me an opportunity to complete this thesis and learn in his laboratory. I am very fortunate to have him as my supervisor, who cared so much about my work and who responded regarding my work and questions very quickly even during his holidays. I am extremely thankful to him for his generosity, advices and guidance. His patience and tolerance towards the deadlines have been greatly appreciated which I will never forget. He provided me an environment and support beyond my expectations. I am also grateful to him for the hope, he created for my career and boosted my confidence level. Without his support, writing this thesis would not possible. I am also sorry to him for sometimes disturbing his busy schedule because of my thesis.
I would also like to thank Prof. Dr. rer. nat. Birger Anspach for agreeing as a second examiner and supporting my extension application as well as his offer to provide the guidance whenever required.
I would like to extend my grateful thanks to the technician of the laboratory of molecular biology and cell culture techniques, Ms. Elisabeth Schäfer, for her valuable supports on cells and instrument handling and general questions in the laboratory. I had enjoyed all the general conversation we did during my experiments. I am also thankful her for teaching me many technical German words.
I must express my gratitude to Bhoomika, my wife, who came here in Germany during the last phase of my thesis and provided me an emotional and mental support. Special thanks to her for making delicious Guajarati food which I never expected to eat here in the foreign country. I am also sorry to her for not giving her enough time which she must deserved.
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Table of Contents
1 Introduction ... 3 1.1 Insect cells ... 3 1.2 Baculovirus biology ... 4 1.2.1 Baculovirus structure ... 4 1.2.2 Baculovirus life cycle ... 4 1.3 Baculovirus expression vector system ... 7 1.3.1 Bac-to-Bac baculovirus expression system ... 8 1.3.2 Green Fluorescent Protein (GFP) ... 10 1.4 Baculovirus amplification ... 11 1.5 Titer determination of baculovirus stocks ... 12 1.6 Aim of the master thesis ... 12 2 Materials and Methods ... 13 2.1 Materials ... 13 2.1.1 Equipment and instruments ... 13 2.1.2 Biological and chemical substances ... 15 2.2 Methods ... 16 Task 1: Expression of higher titer baculovirus in Sf9 cells ... 16 Protocol 1: Thawing Sf9 cells from frozen stock ... 16 Protocol 2: Passaging of Sf9 cells in adherent culture ... 17 Protocol 3: Amplification of baculovirus by infecting Sf9 cells ... 18 Protocol 4: Analysis of baculovirus titer ... 19 Protocol 5: Overlays of images and cell counting ... 21 Task 2: Expression of high GFP concentrations in virus infected Sf9 cells using spinner flask ... 24 Protocol 6: Adapting adherent Sf9 cells to suspension culture and passaging of Sf9 cells in suspension culture ... 24 Protocol 7: Infection of Sf9 cells in suspension culture and protein expression ... 26 Protocol 8: SDS-PAGE and western blot analysis of GFP expression ... 27 3 Results ... 32 3.1 Infection of Sf9 cells with baculovirus ... 34 3.2 Analysis of infection efficiency and titer of the amplified baculovirus using immunofluorescence microscopy ... 35 3.2.1 Fluorescence microscopic analysis of Sf9 cells infected with the amplified baculovirus stocks 35 3.2.2 Estimation of baculovirus titer and Multiplicity of Infection (M.O.I) ... 38 3.3 Determination of the optimal MOI for maximal production of recombinant protein GFP for amplified baculovirus stock P4V2 ... 40 3.3.1 Analysis of GFP expression using amplified baculovirus stock P4V2 by infecting Sf9 cells in spinner flasks with different MOI in serum-free media ... 40 3.3.2 Analysis of GFP expression using amplified baculovirus stock P4V2 by infecting Sf9 cells in spinner flasks with different MOI in the presence of 2 % FCS ... 422 stock P4V2 ... 47 4 Discussion ... 49 4.1 Optimal baculovirus amplification ... 49 4.1.2 Titer estimation of amplified baculovirus ... 50 4.2 Analysis of the factors influencing optimal recombinant protein expression ... 51 4.2.1 Protein expression analysis in serum-free culture condition ... 51 4.2.2 Protein expression analysis in serum-rich culture condition ... 52 4.2.3 Protein degradation ... 53 4.2.4 Defective interfering particles ... 54 5 Summary ... 56 6 References ... 57 6.1 Internet Sources ... 61 7 Appendices ... 62 7.1 List of figures ... 62 7.2 List of tables ... 62 7.3 Abbreviations ... 63 7.3 Statutory declaration ... 65
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1 Introduction
The baculovirus expression vector system (BEVS) is a very effective and popular eukaryotic expression system to produce and express recombinant proteins in insect cells. BEVS can express genes of viruses, fungi, bacteria, and plant in insect cells. In this system, insect cells are infected with baculovirus containing the gene of interest. After infection, the baculovirus regulates the insect cell and inhibits the transcription process of cellular genes until the cell dies. Primary requirements to express the desired protein in BEVS are viable insect cells and a highly infectious recombinant baculovirus stock. To obtain a high titer of baculovirus, many amplification steps are required, which is the most essential process of BEVS, and in all such experimental steps insect cells are needed (Palomares et al., 2014).
1.1 Insect cells
Insect cell culture started with the establishment of the first insect cell lines from the pupal ovarian tissue of the moth Antheraea eucalypti in 1962. Since the establishment of the first insect cell lines, more than five hundred continuous insect cell lines from several species and tissue sources have been established. Among all of these insect cell lines, the three insect cell lines Sf9, Sf21, and
Trichoplusia ni (commercially known as High Five ®) are well defined and most commonly used
for expression of recombinant proteins by BEVS. The Sf21 and Sf9 insect cell lines were originally isolated from the pupal ovarian tissue of the fall armyworm Spodoptera frugiperda, in fact, the Sf9 cell line is a derivative of the Sf21 cell line. The High Five ® cell line derived from the ovarian cell of the cabbage looper Trichoplusia ni. (Druzinec et al., 2013).
In vitro growth curve characteristics of insect cells are relatively similar to the other animal cells.
Insect cells reach an exponential growth phase with doubling times between 18-72 hours after following a short lag phase. Sf9 and Sf21 cells double every 72 and 24 hours, respectively. Doubling time for High Five cells is between 18-24 hours. The optimum temperature range for the cultivation of insect cells is 27 °C to 28 °C, and the optimal pH range is 6.0 to 6.4. Insect cells do not require additional CO2 for growth because of the buffer system used in the media (Agathos et
al., 2010). Insect cells can grow in serum- free media and it is easy to scale up the culture as per
requirement. Insect cell media contain most of the amino acids, trace elements, cholesterol, hormones, vitamins, lipids, and yeastolate. Most serum-free insect cell media also contain additional shear stress protective non-ionic copolymer surfactant Pluronic F-68. Glucose is the most favored carbon source for insect cells, but they also consume fructose and maltose (Palomares
et al., 2014). The oxygen uptake rate (OUR) of insect cells is similar to mammalian cells. On the
other hand, OUR of infected insect cells increases up to 100%, but there is not any increase of other nutrition consumption (Palomares et al., 2005). In this work, Sf9 cells were used during the whole experimental procedure, mentioned in section 2.
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1.2 Baculovirus biology
Baculoviruses are rod-shaped, enveloped, double-stranded DNA viruses with genome sizes of up to 80 to 230 kilobase pairs (Palomares et al., 2005) depending upon virus species. They are pathogenic to insect cells and vastly found in the environment. They have been used in the agriculture industry as a bio-insecticide to control insect populations, but in the field of biotechnology, they are better known as potent protein expression vectors. They are replicated in the nucleus of their host insect cells. They have a very limited host range, mostly insects, so they are safe to vertebrates. The baculoviridae family is classified into four major groups, namely: alphabaculovirus, betabaculovirus, gammabaculovirus, and deltabaculovirus. Baculovirus produces large proteinaceous bodies in their hosts called occlusion bodies (OBs). OBs protect a certain type of virion from the environmental factors. Based on the host species from which the baculovirus was first isolated and the morphology of OBs they form in that species, the
baculoviridae family are further divided into several other prototypes. Among all these prototypes Autographa californica multiple nucleopolyhedroviruses (AcMNPV) is a well-studied and the
most widely used viral vector for BEVS (Au et al., 2013).
1.2.1 Baculovirus structure
The baculovirus produces two types of infectious virus particles: the budded virions (BV) and the occluded-derived virions (ODV). ODV are also known as polyhedra-derived virions (PDV). Both types of virions have different cellular sites of maturation, structural proteins, and infectivity. The nucleocapsid is the central component and is rod-shaped with two distinct ends: apical cap end and a blunt end as shown in figure 1.1. The size of the nucleocapsid measures 30-60 nm in diameter and 250-300 nm in length, which contains a circular, super-coiled, double-stranded DNA genome that encodes 90-180 genes. A major capsid protein is VP39, which has a molecular weight of 39 kDa, and is distributed randomly over the surface of the nucleocapsid. BVs have a plasma membrane-derived envelope, made from the viral fusion proteins GP64 or F protein. ODVs are formed in the nucleus during the late phase of replication. Inside the nucleus, ODVs are incorporated within a protein matrix, mainly polyhedrin forming OBs. ODVs are only infectious when they are released from OBs (Au et al., 2013).
1.2.2 Baculovirus life cycle
The life cycle of baculovirus can be divided into four phases: immediate, early, late, and very late phase shown in figure 1.2. As mentioned above, ODVs are occluded in the OBs (also known as polyhedra) which is made from polyhedrin protein. Polyhedrin is soluble in the alkaline environment (pH>10). In nature, when insect larvae ingest OBs as contaminated food source then the infection cycle begins. During immediate phase (0 to 4 hours post infection), inside the insect gut, OBs are dissolved due to higher pH and ODVs are released from it. Then ODVs infect the midgut epithelial cells. This process is the initiation of the primary infection, and the transcription and translation of viral genes begins. In the early phase (4 to 7 hours after infection), baculovirus
5 DNA replication starts followed by rearrangement of cellular cytoskeleton and degradation of the host genome. During the late phase (7 to 24 hours post infection) individual nucleocapsids bud out from the cell through the cell membrane and acquire an envelope and become BVs.
Figure1.1: Schematic representation of the structure of an infectious virus particle produced by baculovirus upon infection of insect cells. Budded virions (BV) and occluded derived virions (ODV) both contain several proteins in their nucleocapsid. Major capsid protein for both virions is VP 39 and it is present in the entire nucleocapsid. VP78/83 are assumed to locate in the blunt end of the nucleocapsid. BV envelope contains the fusion protein GP64 or F protein which forms peplomers on one end. GP64 and F protein belong to alphabaculoviruses subclass of nucleopolyhedrovirus (NPV) group 1 and 2, respectively (Au et
al., 2013).
These budded virions cause the secondary infection to neighbour cells and tissue and spread the infection throughout the insects. In the very late phase (24 -120 hours post infection), the production of polyhedrin (33 kDa) and p10 (10 kDa) protein starts and virions accumulated in the nucleous are occluded into polyhedra and form OBs. Formation of OBs continues until the insect disintegrates and after that OBs are released and ingested by other insects (Williams et al., 2017; Palomares et al., 2015). The polyhedrin can be seen in the nuclei of infected cells. The p10 protein is associated with nuclear and cytoplasmic fibrillar structure in infected cells (Chaabihi et al., 1993)
The secondary infection and systemic spread are done only by BVs. In the culture of insect cells
in vitro, only BVs cause the infection. The infection cycle of BVs in cultured cells is shown in
figure 1.3. First, BVs enter into the cell through endocytosis. The fusion protein on its envelope like GP64 binds with the cellular receptor of the cells. During the acidification of the endosome,
6 the endosomal membrane fuses with the viral envelope, causing the nucleocapsid to be released into the cytoplasm.
Figure1.2: Schematic representation of in vivo life cycle of baculovirus. The life cycle of baculovirus is divided into four phases. Immediate phase: Ingested polyhedra is solubilized in the insect midgut due to alkaline environment, which leads to the release of the occluded derived virions (ODV). These ODV initiate the primary infection by infecting epithelial cells. Virions migrate to the cell nucleous, and the transcription and translation of viral genes starts. Early phase: the cellular cytoskeleton and nucleous are rearranged, the host genome degradation occurs and replication of baculovirus DNA starts. In the late phase, nucleocapsids bud through cell membrane and acquire an envelope and become budded virus (BV). BV cause the systemic spread throughout insect during the secondary infection. During the very late phase, production of polyhedrin and p10 genes begin. Virions accumulate in the nucleous and are occluded into polyhedra. Polyhedra crystals accumulate till the insect dies and disintegrates. After that polyhedra crystal are released and consumed by other insects (Palomares et al., 2015).
These nucleocapsids are then transported towards the nucleus. Actin polymerization is used for the trafficking of nucleocapsids towards the nucleus. Then, the viral DNA is released into the nucleus and the replication process begins by transcription of essential genes in the center of nuclei of infected cells which is the cellular location of the nucleocapsid assembly. Progeny nucleocapsids leave the nucleus and acquires an envelope from the nuclear membrane and move towards the plasma membrane. In the cytoplasm, they acquire a glycoprotein-rich envelope and bud off from the cell membrane and form BVs. These BVs infect neighbouring cells, and the infection cycle starts again (Au et al., 2013).
7 Figure1.3: Schematic representation of budded virus infection cycle in cultured cells. In step 1 budded virus enters into tissue culture cells through endocytosis. Step 2: due to acidification, the nucleocapsid releases into cytoplasm and is transported towards the nucleus using actin polymerization. Step 3: release of the viral DNA into the nucleus and viral replication starts with the formation of the virogenic stroma near the center of the nuclei of the infected cells. At this site viral DNA is transcribed and replicated as well as nucleocapsid is assembled. Step 4: progeny nucleocapsids enter the nucleus and acquire an envelope from the nuclear membranes that is eventually lost. Step 5: nucleocapsids move towards the plasma membrane where they bud off acquiring a glycoprotein-rich envelope from the cell membrane to generate budded virions. These budded virions cause systemic infection by infecting neighboring cells and the cycle of infection starts again. (Au et al., 2013)
1.3 Baculovirus expression vector system
There are two characteristics of baculoviruses which make them attractive for the use as an expression system. The first is that the very late p10 and polyhedrin genes are not required to produce BVs, the type of virus which is responsible for the systemic infection in insect cells. The second is that promoters of both virus genes are very strong and if they are fused to the coding region of any foreign gene then they have the potential to produce large amounts of recombinant protein in insect cells. These characteristics of baculovirus were the basis of BVES. It is also possible to generate expression vectors that contain multiple copies of these promoters so that
8 recombinant proteins can be produced together. Insect cell lines are additionally capable in promoting post-translational modifications required when producing more complex proteins from eukaryotic cells.(Kelly et al., 2016)
The baculovirus has a considerably large genome size for that reason integration of the foreign gene by direct cloning is relatively difficult. In most systems, the foreign gene is cloned into a plasmid or transfer vector. However, one commercial expression system (BaculoDirect TM from Thermo Fischer Scientific) nowadays inserts the foreign gene directly into the linear virus DNA via Gateway ® recombination technology. The classical approach to produce recombinant virus in BEVS is by homologous recombination between the viral genome and transfer vector containing the foreign gene under the control of p10 or polh promoter and by transfection into the host insect cells. The homologous recombination occurs when the foreign gene is inserted into virus DNA. After insertion, the polyhedrin gene is not functional anymore, so this virus genome replicates and produces recombinant budded virus without the formation of OBs. This budded virus is then harvested from the culture medium and the foreign gene expression is analyzed. This approach was first reported for the expression of human beta interferon in 1983 (Smith et al., 1983). The homologous recombination technique required a separation of the recombinant virus from the parental virus, which was done by plaque assay and plaque purification methods. However, these methods were very complex and difficult even for experienced users and also took more than one month to complete. Several other approaches have been tried by modifying virus genome and the transfer vector effectively. The most rapid and efficient method to produce the recombinant baculovirus was developed by the introduction of the bacmid technology, which no longer required to rely on plaque assay for the separation and purification of recombinant virus. Today many different baculovirus expression systems available are based on the bacmid technology. The baculovirus used in this work was generated by the Bac-to-Bac ® (Thermo Fisher Scientific) baculovirus expression system. (King et al., 2016)
1.3.1 Bac-to-Bac baculovirus expression system
The Bac-to-Bac system is based on the site-specific transposition of the foreign gene to be expressed from the baculovirus shuttle vectors, maintained as a bacmid in E. coli cells. The main requirements of this system are a (a) pFastBacTM series donor plasmid into which the gene of interest is cloned and (b) an E. coli host strain DH10BacTM used as a host for the donor plasmid. The pFastBacTM donor plasmid consists of a multiple cloning site, gentamicin resistance, ampicillin resistance, and a SV40 polyadenylation signal inserted between left and right arms of Tn7 elements as shown in figure 1.4. A series of pFastBacTM vectors is available, thus one has to choose the optimal vector based on the experimental requirement. In this work, pFastBacTMHT is used which has N-terminal 6 x His-tag for the purifying recombinant protein. DH10BacTM contains low copy number mini F replicon, kanamycin resistance marker and a segment of DNA encoding LacZa peptide.
9 Figure1.4: Schematic representation of the pFastBacTM HT donor plasmid. This plasmid contains polyhedrin promoter site, ampicillin and gentamicin resistance genes, origin of replication site, position of Tn7 elements and multiple cloning site with N-terminal 6 x His-tag. (source: Noi et al., 2016)
To generate recombinant baculovirus with the Bac-to-Bac expression system, the gene of interest was first cloned into the multiple cloning site of the pFastBacTM HT donor plasmid downstream and under the control of the baculovirus specific promoter, derived from polyhedrin gene. After successful integration, the recombinant donor plasmid was transformed in E. coli strain DH10BacTM, which contains the baculovirus genome as a bacmid. This site-specific transposition produces the recombinant bacmid DNA. During the transposition process, cells are selected using the antibiotic selection factor. Transposition occurs between mini Tn7 attachment site on the bacmid DNA and mini Tn7 elements on the pFastBac plasmid. This transposition disrupts the expression of LacZa peptide and generates the recombinant bacmid (Anderson et al., 1995). The recombination colonies are identified based on blue or white colour selection. The colonies containing the recombinant bacmid are white, which are then isolated by plating onto selective media and resulting in bacmid DNA that can be used to transfect insect cells for virus production. The culture media collected from the transfected cells contains the first generation of recombinant baculovirus stock (P1 stock). The schematic diagram of the Bac-to-Bac expression system is shown in figure 1.5. This P1 virus stock used to infect insect cells or to determine the virus titer. Further amplification of baculoviruses can be obtained by infecting insect cells with this P1 virus stock. Green Fluorescent Protein (GFP) is used as a gene of interest for this work (King et al., 2016; Invitrogen, 2015).
10 Figure1. 5: Schematic representation of the generation of the recombinant baculovirus with Bac-to-Bac expression system. The first step consists of the cloning of the gene of interest into the recombinant donor plasmid. This plasmid is transformed into competent DH10Bac E. coli cells where the integration into the bacmid takes place. After using an antibiotic selection factor, the transformed cells can be isolated and the recombinant bacmid can be purified via Mini-prep. This recombinant bacmid used to transfect the insect cells for the production of recombinant baculovirus particles. The medium containing the recombinant baculovirus particle is used either to determine the titer of the baculovirus stocks or to infect other insect cells for the recombinant gene expression and further amplification of the virus stocks. (Source:www.thermofisher.com)
1.3.2 Green Fluorescent Protein (GFP)
The protein GFP (Green Fluorescent Protein) consists of 238 amino acids and has a molecular weight of 27 kDa. It was first isolated from the jellyfish aequorea victoria in 1962. The cDNA of GFP was cloned from the jellyfish in 1992. GFP is a well-recognized fluorescent based reporter gene and used for the expression and localization of fusion proteins in the biological and medical research. In nature, GFP emits green light when exposed to ultraviolet light or simply blue light because of the transfer of energy from protein aequorin to GFP. GFP has a major absorption peak at 394 nm and a minor peak at 470 nm. Its emission peak is at 509 nm. GFP is the species-independent fluorescent reporter; it does not require any substrate, additional protein, or cofactor for its detection. Moreover, GFP also lacks the pretreatment for detection, which are often toxic to the cells. Many small fluorescent molecules like FITC (Fluorescein isothiocyanate) are toxic to the live cells, but GFP is much less harmful when illuminated in living cells. GFP can be easily
11 detected under a fluorescence microscope (Wu et al., 2016). The GFP has a beta barrel structure (shown in figure 1.6) made up of 11 beta strands with alpha helices containing the covalently bonded chromophore running through the center. These alpha helices form caps on the end of the structure and the chromophore is attached to them and buried. This barrel structure is also known as beta can and is unique to the GFP family (Tsien, 1998).
Figure1.6: Schematic representation of the structure of aequorea victoria Green Fluorescence Protein (GFP). GFP has 11 beta sheets (green colored) forming a beta barrel like structure which is threaded with an alpha helix (yellow) bearing chromophore, shown in ball-and-stick representation in the center. Approximate length and diameter of this beta barrel structure are 4 nm and 3 nm, respectively. (source: www.studocu.com)
1.4 Baculovirus amplification
The amplification of the baculovirus to high titer stocks is one of the most significant and critical parts of the whole process because low-quality virus stocks contribute to very low productivity. There are two major problems that can affect the quality of baculovirus stock. The first is genetic instability and the second is defective interfering particles (DIP). For the former problem, the gene of interest (GOI) is lost during the amplification of virus stock by subsequent passaging. To overcome this issue, the vector should be designed carefully to avoid an unstable construction. The second problem DIP is the appearance of mutant virus particles during the passaging of virus stock, which alter the infection process (Palomares et al., 2005). DIPs are usually not able to form plaques, and morphologically they are smaller in size compared to average normal viral particle size. Genetic deletion in DIPs is believed to be nearly 43% of the total genome length.
Without the presence of the complete virus, DIPs are unable to replicate themselves. They require missing genes for the replication. This is the primary reason that the multiplicity of infection (MOI) plays a vital role in the propagation of baculovirus stocks. The MOI refers as the ratio of the number of infectious virus particles added to the number of cells in the culture. It is expressed as
12 plaque forming units (pfu) per cell. At high MOI, there is a high probability that DIPs and intact virus can enter into the same cell. In that case, both will compete for the components of the replication process. DIPs can replicate faster than the normal viruses. Therefore, the yield of infectious virus decreased. To reduce the appearance of DIPs, it is recommended that the amplification of baculovirus stock should be performed at low MOI (≤ 0.1). Thus, infection of cells with more than one virus particle becomes an unlikely situation. Furthermore, the passage number of the baculovirus stocks should be kept as low as possible, ideally less than 6 because prolonged passage promotes the accumulation of DIPs. DIPs replicate at the expense of intact baculovirus and cause low amount of infectious virus and protein production losses. New baculovirus stock should be generated from low passage stocks upon detection of genetic instability or DIPs. Either Sf9 or Sf21 cell lines should be used for the generation and amplification of baculovirus stocks (Palomares et al., 2015; Oers et al., 2015).
1.5 Titer determination of baculovirus stocks
It is very essential to know the titer of baculovirus stocks prior to preparing new virus stock and /or infecting cells for protein production experiments. Precise determination of the baculovirus titer is a very challenging and critical task. The optimum production of recombinant protein requires the knowledge of virus titer and cell viability. Incubation time and cell concentration are significant parameters which affect the titer of baculovirus. Traditionally, two methods, plaque assay and end point dilution are used most commonly determine the baculovirus titer. Both methods directly measure the infectivity of baculovirus in cells. Although the plaque assay determines the virus titer more accurate than the other method, it is technically challenging and takes nearly two weeks. Moreover, to identify the plaque demands an experienced person. On the other hand, the endpoint dilution method is easy to perform but it is difficult to interpret the results. (Roldao et al., 2009). After titer estimation the baculovirus stock should be stored at 4°C for a short time and protected from light or it can be stored at -70°C or in liquid nitrogen for long term storage. Virus stock should be titered again after the long-term storage because it will lose activity over time. It is also common practice to add fetal bovine serum (FBS) to virus stocks to increase their stability. In this work, a different approach for titration is used, the expression of the green fluorescent protein (GFP) is followed as a reporter to estimate the titer of baculovirus.
1.6 Aim of the master thesis
The motivation for this master thesis initiated from the result of a master project performed by Ghanemi and Hmaid in the laboratory of molecular biology and cell culture techniques, HAW Hamburg. They observed that infecting Sf9 cells with low baculovirus concentration leads to similar amplification rates as with ten times as much virus when the time for virus production is prolonged from 96 to 120 hours. Based on their findings this master thesis has two aims. The first aim is to establish experimental conditions for maximal virus amplification using only little amounts of virus. The second aim is to determine for the amplified virus the optimal MOI for maximal production of recombinant protein. In order to follow virus amplification and protein expression, a baculovirus containing the gene for the reporter protein GFP is used.
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2 Materials and Methods
2.1 Materials
2.1.1 Equipment and instruments
Equipment / Instrument Manufacturer / Distributor, Model
Automated cell counter CountessTM, Invitrogen
Laminar flow hood Hera safe, Model -KS18
CO2 incubator Binder
Tube rotator European Molecular Biology Laboratory
Magnetic stirrer plates Thermo Fisher Scientific Inc.
Inverted microscope Carl Zeiss AG, Axiovert 40 CSL
Microscope camera Olympus DP71
Water bath Gesellschaft für Labortechnik GmbH
Centrifuge (large) Multifuge 3 S-R, Heraeus Holding GmbH
Centrifuge (small) Mini-spin Eppendorf
Fluorescence microscope Olympus BX41
Magnetic stirrer IKA®-Werke GmbH, Big-squid
Cell scraper Falcon, 18 c.m handle and 1.8 c.m blade
Conical tube (15ml ,50ml) Flacon
24-well cell culture plate (sterile) CellStar, cat No. 662160, Greiner Bio-one
Glass coverslips Thermo Scientific, Germany
Tissue culture flasks (T-75, T-25) CellStar®, Greiner Bio-one GmbH
Spinner flasks (250 ml) Bellco Biotechnology, USA
Micro pipettes Eppendorf AG, (P10, P20, P100, P1000)
Pipette tips (white, yellow, blue) Eppendorf AG
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Digital camera Olympus Corporation
Electrophoresis power supply-EPS 601 GE Healthcare
Refrigerator (4 oC) Liebherr-International AG
Freezer (-20 oC) Liebherr-International AG
FUSION-FX7 Advance-Multi-Imaging
Vilber Lourmat Deutschland GmbH Laboratory glassware washer Miele, Inc.
Mini Trans-Blot electrophoretic transfer cell Bio-Rad Laboratories, Inc. Trans-Blot system with plate electrodes Bio-Rad Laboratories, Inc. Mini-PROTEAN Tetra Cell Bio-Rad Laboratories, Inc. Blotting filter papers Bio-Rad Laboratories, Inc.
Rotating platform Labortechnik Fröbel GmbH
Microscope slide Carl Roth GmbH
Reaction tubes (1 mL, 2 mL) Eppendorf AG
Serological pipette (sterile),1,2,5,10 and 25ml CellStar®, Greiner Bio-one GmbH
Fine tweezers Carl Roth GmbH
Electronic weighing scales Sartorius Mechatronics
Glass petri dish Thermo Fisher Scientific Inc.
Laboratory glassware Miele, Inc.
Vortex mixer Heidolph Reax Top
Digital heat block Eppendorf AG
Measuring cylinder Miele, Inc.
Laboratory trays European Molecular Biology Laboratory
Heat sealer Severin Electrogerät GmbH
Casting chamber Bio-Rad Laboratories, Inc.
Casting stand Bio-Rad Laboratories, Inc.
Glass plates and spacer Bio-Rad Laboratories, Inc.
Rocking table European Molecular Biology Laboratory
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2.1.2 Biological and chemical substances
Biological and chemical substances Manufacturer / Distributor
Sf9 cells (Passage 7) acCELerate GmbH, Hamburg
Recombinant Baculovirus (P3) stock, containing GFP gene and His6-tag
Prepared as per Thermo Fisher Scientific protocol
Serum-free cell media Ex-cell ® 420, with L-glutamine Penicillin (1000 U/ml) Thermo Fisher Scientific Inc. Streptomycin (1000 U/ml) Thermo Fisher Scientific Inc.
Fetal Calf serum PAN-Biotech, Cat.- No.: P40-37500
Protease-Inhibitor cocktail (97% DMSO) Sigma-Aldrich Co. LLC. Novex Sharp Pre-stained Protein Standard Thermo Fisher Scientific Inc. IRdye 800CW antibody Li-cor, Cat.-No.: 926-3221110.2015 anti-His mouse antibody Dianova, Cat.- No.: DIA-900 Rotiphorese Gel 30 (37.5:1): 30 %
Carl Roth GmbH Ethanol denatured ≥ 98 %, Carl Roth GmbH
DL-Dithiothreitol Sigma-Aldrich Co. LLC.
Ammonium persulphate (APS) Carl Roth GmbH Dimethyl sulfoxide (DMSO) Carl Roth GmbH
Formaldehyde solution Carl Roth GmbH
Methanol ≥ 99% Carl Roth GmbH
N,N,N',N'-Tetramethyl-ethylenediamine
Carl Roth GmbH
Tween 20 GE Healthcare
DAPI Sigma-Aldrich Co. LLC
Mowiol Calbiochem, USA
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2.2 Methods
Task 1: Expression of higher titer baculovirus in Sf9 cells
Protocol 1: Thawing Sf9 cells from frozen stock
Below is mentioned material and equipment required to culture the Sf9 cells. Additional protocol specific materials are also mentioned with each protocol. All materials with manufacturer name also mentioned separately in section 2.1.
General material list:
Ø Sf9 cells (acCELLerate GmbH, passage 5 obtained from lab assistant Elisabeth Schäfer) Ø T-75 flasks
Ø CO2-incubator capable of maintaining 27 ℃ ± 0.5 ℃ (with 0.0% CO2) Ø 27°C water bath
Ø Sf9 cell medium (Ex-cell ® 420, with L-glutamine) Ø Centrifuge
Ø Laminar flow hood suitable for cell culture
Ø Penicillin (10000 U/ml) & streptomycin (10000 U/ml) added to the media (5ml per 500 ml media)
Ø Conical tubes 15 ml, 50 ml Ø Pipet aid, automated Ø Fatal Calf Serum (FCS) Ø Ethanol (70%)
Ø Serological pipette, (sterile): 5, 10 and 25 ml volumes
(Note: In a case of using of Fatal Calf Serum (FCS), add 2% of prewarmed FCS aseptically with a pipette to the medium under the laminar flow hood)
1. Place the bottle of media in a water bath at 27°C for 30 minutes. Retrieve frozen ampoule of Sf9 cells from liquid N2 and immediately transfer it to the water bath at 27°C.
2. Gently agitate ampoule until cells are almost thawed, then quickly sterilize the outer surface of the ampoule with 70% ethanol and transfer to laminar flow hood.
3. Using aseptic technique in the laminar flow hood, carefully open ampoule. Slowly pipette contents of the ampoule into a sterile 50 ml polypropylene centrifuge tube.
4. Add 10 ml pre-warmed Sf9-medium dropwise to cells in the centrifuge tube and gently swirl the centrifuge tube at the same time.
17 5. Close the cap of the centrifuge tube and centrifuge it at 80 x g for 4 minutes at room
temperature.
6. Bring the centrifuge tube back to the laminar hood and remove supernatant aseptically with a pipette.
7. Add 10 ml of pre-warmed fresh medium dropwise to the cell pellet in the centrifuge tube and meanwhile gently swirl it.
8. Add 10 ml of pre-warmed fresh medium to new a T-75 flask. Gently pipette cell suspension in centrifuge tube 3–5 times. Transfer entire cell suspension into the T-75 flask.
9. Gently rock the T-75 flask to ensure the even distribution of cell suspension on the bottom surface and label the date, passage number, and personal initials on the T-75 flask.
10. Incubate T-75 flask at 27 °C in an incubator for cell culture (CO2 not required) until monolayer becomes 80-90% confluent and observe the cells on a daily basis with a phase-contrast microscope.
Protocol 2: Passaging of Sf9 cells in adherent culture
Material list:
Ø Inverted and phase contrast light microscope with digital camera Ø Cell scraper
Ø Sf9 cell medium (Ex-cell ® 420, with L-glutamine)
1. Examine the Sf9 cells grown in a 27 °C incubator as adherent culture using a phase-contrast microscope to ensure that cells are confluent (>80 %), healthy and not contaminated. If required, take a picture of cells with a 100 x magnification for documentation purposes. 2. Transfer pre-warmed medium (27 °C), flask containing cells, new flasks, pipettes, and cell
scraper into the laminar flow hood.
3. Add desired amount of fresh medium (T-25 flask = 5 ml, T-75 flask = 15 ml) into each new flask.
18 5. Add desired amount of fresh medium (T-25 flask = 5 ml, T-75 flask = 10 ml) into confluent flask. Detach cells with the help of a cell scraper by tilting the flask on a 45º angle and streaming the medium over the cells on the plastic surface, passing flow back and forth. Repeat until all cells are removed from the entire bottom of the flask.
6. Aspirate detached cells with the 10 ml pipette and transfer half of the cell suspension into two new flasks of the same size for a 1:2 dilution.
7. Label the flasks with cell line name, date, passage number, dilution factor, initials of the person and put them into the incubator to let them grow at 27 °C without CO2 for three days. Observe the cells with a phase-contrast microscope on a daily basis.
8. Whenever the cells cover the bottom of the flask (reach 80 -100 % confluency), they should be passaged again into new flasks.
Protocol 3: Amplification of baculovirus by infecting Sf9 cells
Material list:
Ø Recombinant baculovirus stock (recombinant for GFP, passage 3, obtained from lab assistant Elisabeth Schäfer)
1. Prepare three of subconfluent Sf9 cells (60 -70 % confluent) of T-25 flasks. Label the flasks as 1, 2 and 3. Use two flasks for virus amplification and one flask as a negative control. Take a picture of uninfected cells with a phase-contrast microscope with the 100x magnification lens.
2. Aspirate the spent medium from all three flasks and add 5 ml 27 °C pre-warmed fresh medium to each flask.
3. Add 20 μl and 40 μl of P3 recombinant baculovirus stock in flask 1 and flask 2, respectively. Let flask 3 uninoculated and transfer all three flasks in the incubator at 27 °C for 120 hours.
4. After 120 hours post-infection, observe the cells under phase contrast microscope and take pictures with a 100 x magnification.
5. Collect the medium containing the amplified virus from flask 1 and flask 2 in two separate 15 ml centrifugation tubes.
6. Centrifuge both tubes at 500 x g for 5 minutes at room temperature and collect the supernatant aseptically under laminar flow hood with a 10 ml pipette and transfer it in two separate conical tubes. This material corresponds to the amplified baculovirus P4 stock.
19 7. Label the conical tubes containing the supernatant from flask 1 and flask 2 as baculovirus stock P4V1 and P4V2 respectively and store them in the refrigerator at 4 °C and protect from light by wrapping with aluminum foil.
8. Analyse titer of both virus stocks as described in protocol 4 and protocol 5 then produce a larger volume of high titer virus stock in T-75 flasks by scaling up all the parameters accordingly.
Protocol 4: Analysis of baculovirus titer
Material list:
➢ Glass coverslips
➢ 24-well cell culture plate (sterile)
➢ Automated cell counter
➢ 0.4 % trypan blue diluted in 1x PBS -➢ Fine tweezer
➢ Tube rotator ➢ Filter paper
➢ Cell counting chamber slides ➢ Pipette tips
➢ Glass petri dish
➢ Fluorescence microscope
➢ 1x Phosphate Buffered Saline (PBS-)
➢ Diamidino-2-phenylindole (DAPI) dye - 1: 10,000 diluted 1x PBS
-➢ 3 % Paraformaldehyde prepared in 1x PBS
-➢ 50 mM NH4Cl prepared in 1x PBS-: Dissolve 0.1 g NH4Cl in 50 ml of 1x PBS- at room temperature
1. Prepare glass coverslips for seeding cells. For fluorescence microscopic analysis it is necessary to grow cells on thin glass coverslips. For that place glass coverslip in a conical tube containing 70 % ethanol on tube rotator for 24 hours. Rinse each coverslip with distilled ddH2O. Dry the coverslips and autoclave them in a glass petri dish between the layers of filter paper.
2. Insert a single clean and dry glass coverslip with sterile tweezer into each well of a 24-well plate, which will be used to seed the cells and cover the plate with the lid.
20 3. Detach the confluent Sf9 cells growing in culture flask using a cell scraper and transfer the
cell suspension into a falcon tube.
4. Count the cells using automated cell counter. To count the cells, add 50μl of the cell suspension to 50 μl of 0.4 % trypan blue stain. Mix gently by pipetting up and down. Cut off the first 2 mm of a yellow pipette tip before using it for the cell suspension to avoid cell breakage. Add 10μl of the mixture to the chamber ports on one side of the chamber slide. 5. Insert the Countess ™cell counting chamber slide into the counter port and press the
“Count Cells” button. Note the total cells, viable cells, dead cells, and viability numbers. 6. Dilute the cell suspension accordingly so that to each well of 24-well plate 1 x 105 cells (in
1ml medium) is pipetted. Resuspend the cell suspension by pipetting up and down gently 2-3 times prior to seed them in 24-well plate.
7. Push the glass coverslips of each well with sterile tip very gently to the bottom of the well so that it remains in close contact to the bottom surface of the well and avoid the attachment of the cells on the backside of the glass coverslips.
8. Put the 24-well plate into the incubator and grow the cells for 24 hours at 27 °C.
9. After 24 hours, infect the cells of six wells with volumes of 10 μl, 25 μl and 50 μl of the amplified baculovirus stocks, obtained from protocol 3, either from the P4V1-virus stock or P4V2-virus stock, respectively. Make sure that to all six wells the appropriate volume of virus stock from the either of virus stock is added. Perform the experiment in duplicates and leave one well uninoculated as a control well.
10. Put the 24-well plate into the incubator and let the virus infect the cells for 48 hours at 27 °C.
11. After 48 hours perform PFA-fixation and fluorescence microscopic analysis by following the steps mentioned below. Make sure to work fast for any exchange of liquids, do not let the cells dry for more than 20 seconds during the following procedure. All steps are performed at room temperature.
12. Aspirate the medium from the wells and wash the cells once for 3 min with 1ml of 1x PBS -13. Use paraformaldehyde as a fixative and fix the cells in 3 % paraformaldehyde solution. For
that aspirate the PBS- from each well and add 0.5 ml of 3 % paraformaldehyde solution for 30 min in each well, then wash cells 2 x 5 min with 1x PBS-.
14. In order to quench the aldehyde groups, remove PBS- and add 1 ml of 50 mM NH4Cl in
21 15. For staining of DNA in the cell nuclei remove PBS- and add 0.5 ml of diluted DAPI solution
(1: 10,000 in 1x PBS- diluted) in each well for 2 min. DAPI solution is carcinogenic. Wear
hand gloves and make sure to avoid its contact with the skin.
16. For mounting cells, place 5 μl of Mowiol on a microscope slide. Remove the glass coverslips from the well with tweezers and dip the glass coverslip with the cells briefly in ddH2O, remove the excess ddH2O on a kleenex cloth and embed cells on glass coverslip
with cells oriented towards the drop of mowiol on the microscope slide. Allow to dry them for 30 minutes at 37 °C in the incubator or overnight at room temperature in the drawer, in the absence of light. Analyse all the samples under the fluorescence microscope.
17. Place the dry microscope slide with glass coverslips that contain the cells under a fluorescence microscope with 100 x magnification. Observe each coverslip one by one and select the best region on the coverslips where the cells are evenly distributed. Calculate the total surface area of coverslip for the total cell number calculation.
18. Take two photos of the selected region with first the filter setting DAPI for total cells (all nuclei stained) and second FITC for baculovirus infected cells expressing GFP with exposure times of approximately 90 ms and 1400 ms, respectively.
19. Save the blue (DAPI) and green (GFP) images as TIF files and make overlays of these images using Adobe Photoshop software. Count the total cells stained with DAPI versus green-colored cells expressing GFP which represent virus infected cells.
Protocol 5: Overlays of images and cell counting
1. Open both saved images of the selected area captured as described in protocol 4 by using sequentially DAPI filter (blue) and FITC (green) filter in adobe photoshop on a computer. 2. Choose “RGB color” and “16 bit/Channel” by clicking Image > Mode> RGB color, 16
Bits/ Channel for both the images.
3. Open new file by File > New (or Ctrl + N). A new window will appear, select “RGB color” and “16 bit/Channel” and choose the background “white” in that window for the new file. Name this file as “overlay”.
4. For the blue image select only blue channel and for green image select only green channel from the bottom right corner of the adobe photoshop screen.
22 5. Activate blue image and select whole image by clicking Image > Select > All (or Ctrl +A) and copy it by clicking on Edit > Copy (or Ctrl C) then go back to “overlay” file and select only blue channel from the bottom right corner and paste copied blue image via Edit > Paste (or Ctrl V). Similarly, copy green image but select only green channel in “overlay” file and paste it.
6. Fill black color as a background in red channel by clicking Edit > fill > background colour > (or Shift +f5) and select black colour as background. Now select all the channels from the bottom corner and adjust the colour of the image by Image > Adjustments > Levels and small window will appear as shown in the figure 2.1 choose Auto and then if required, adjust brightness, contrast and tonal range of the image by adjusting the grey triangle accordingly and save the image.
Figure 2.1: Adjustment of colour and contrast in Adobe Photoshop. By clicking Image > Adjustments >Level, this window will appear. Adjust the colour and contrast by choosing Auto and adjusting the grey triangle shown in the middle. (Screenshot is taken from Photoshop of German version)
7. For counting the cell numbers, select the 25 % of the picture are with RGB channel with the help of horizontal and vertical measurement ruler. Copy the selected area and paste it in the new window.
8. For the counting green cells, select the green and red channel so that green cells will appear with black background shown in Figure 2.2. Similarly, for the counting of blue cells select the blue and red channels so that blue cells appear with black background shown in figure 2.3.
23 9. Begin the counting of the cells select “Count Tool” via Analysis > Count Tool so that the following options available, as shown in figure 2.4. With the help of mouse count each cell by clicking on it one by one.
Figure 2.2: Screenshot of DAPI filter image opened in Adobe Photoshop. Image shown is 25 % of the whole image. In this image nuclei of the cells shown in blue which are counted for the estimation of total cell number. Count of the cells are shown with white marker.
Figure 2.3: Screenshot of FITC filter image opened in Adobe Photoshop. Image shown is 25 % of the whole image. In image infected cells are shown in green. Count of the cells are shown with white marker.
24 10. Note the numbers of blue (total cells) and green (infected cells) cells of each image and use these numbers for further calculations of virus titer estimation.
Figure 2.4: Different functions of Count Tool.
Task 2: Expression of high GFP concentrations in virus infected Sf9
cells using spinner flask
Protocol 6: Adapting adherent Sf9 cells to suspension culture and passaging of Sf9
cells in suspension culture
(Note: In case of using Fatal Calf Serum (FCS), add 2% of prewarmed FCS aseptically with a pipette to the medium under the laminar flow hood)
Material list:
➢ 250 ml spinner flasks ➢ Magnetic stirrer plates
25 ➢ Countess ™ cell counting chamber slides
➢ 0.4 % trypan blue stain
➢ Sf9 cell medium (Ex-cell ® 420, with L-glutamine) ➢ Fatal Calf Serum (FCS)
➢ Penicillin (10000 U/ml) & streptomycin (10000 U/ml) added to the media (5 ml per 500 ml media)
1. Prepare 3-4 confluent T-75 flasks of adherent Sf9 cells, detach them as described in protocol 2 and transfer the cell suspension into conical tubes.
2. Resuspend the settled cell suspension by pipetting 2-3 times up and down and remove 100 μl of cell suspension as a sample for cell counting in 1.5 ml sterile Eppendorf tube.
3. Count cells with automated cell counter as described in protocol 4 step 4-5. Collect data for total cells, live cells, dead cells, and viability of the sample. The viability of the cells must be more than 90 % prior to start the cultivation in a spinner flask.
4. Based on total cell number, inoculate and dilute the cell suspension with fresh culture media in a manner that the spinner flask contains 5 x 105 – 1 x106 cells/ml and total volume of the cell suspension should be 50 ml.
5. Loose both arm caps of spinner flask by one full turn to allow proper gas exchange. Incubate culture at room temperature (≅22 °C) on a magnetic stirrer plate with constant stirrer at the rate of 70 – 80 rpm and allow them to grow.
6. After 24 hours, remove spinner flask from the magnetic stirrer plate and take a small sample from the spinner flask using a sterile 2ml pipette under the laminar hood. If cells have settled before taking the sample, swirl the flask to distribute the cells in the medium evenly.
7. From the sample, determine the total number of cells and percentage of viability using the Countess automated cell counter. Calculate the volume of media that needed to add to dilute the culture to a density of 5 x 105-1 x 106 cells/ml.
8. Aseptically add the appropriate volume of pre-warmed medium into the spinner flask or split the cell suspension to multiple flasks if needed. Continue culturing cells on the magnetic stirrer.
26
Protocol 7: Infection of Sf9 cells in suspension culture and protein expression
Material list:
➢ Amplified baculovirus stock (p4); obtained as described in protocol 3 ➢ 2 x SDS sample buffer:
Add 100 mM Tris-HCl pH 6.8 (1.21 g Trizma Base / 100ml), 20 % glycerol (20 ml / 100 ml), 4 % SDS (4 g / 100 ml), 0.02 % bromophenol blue (20 mg / 100 ml) in beaker and make up to 100 ml with ddH2O, filter with 0.45 μM filter, keep at room temperature. Add 100 mM DTT to the final concentrations shortly before use to reduce the disulfide bonds in the sample.
1. Prepare 10-12 T-75 flasks of confluent Sf9 cells required to expand them in four spinner flasks. Detach the cells from the flasks and prepare all four spinner flasks with Sf9 cell suspension as described in protocol 6. Label the spinner flasks as 1,2,3 and 4.
2. When the Sf9 cells in all four spinner flasks have reached a concentration of 1-2 x 106 cells/ml, infect the cells with three different M.O.I of amplified recombinant baculovirus stock (P4) obtained as described in protocol 3.
3. For infection of cells, add 1 ml, 5 ml, 10 ml of P4 baculovirus stock in spinner flask 1, 2, and 3, respectively. Maintain spinner flask 4 uninfected as control. Incubate all spinner flasks on a magnetic stirrer at 70 rpm for five days at room temperature.
4. Collect a 1ml sample from each spinner flask at 0, 24, 48, 72, and 96 hours post infection in a small 1.5 ml Eppendorf tube. Use 50 μl of the sample from each day and from each spinner flask to determine the total, live, and dead cell numbers along with percentage of cell viability. Compare cell morphologies and cell densities of infected cell cultures with non-infected controls to confirm progress of the infection.
5. Centrifuge aliquots of cell suspension that were transferred to 1.5 ml sterile Eppendorf tubes from each spinner flask on each day, which contains 1 x 106 cells at the speed 80 x g for four minutes at room temperature.
6. Remove supernatant and resuspend pellet in 25 μl of 1 × SDS sample buffer by gently pipetting up and down and avoid foaming.
7. Samples are boiled for 5 min at 95 °C in a heating block and stored frozen at -20 °C until analysis of all samples by SDS-PAGE and western blotting for GFP-expression.
27
Protocol 8: SDS-PAGE and western blot analysis of GFP expression
Materials list:
Ø Short plate Ø Spacer plate
Ø Electrode assembly with banana plug Ø Casting stand and casting frame Ø Mini tank and lid
Ø Inner chamber Ø Plastic comb Ø Clamping frame
Ø Protein molecular weight standard - “Novex® Sharp pre-stained protein standard” Lot No. 1022458
Ø 30 % Acrylamide/0.8 % Bisacrylamide stored at 4 °C -(Carl ROTH Rotiphorese Gel 30 (37.5:1) Concentration 1.024 g/ml)
Ø 1 M Tris-HCl pH 6.8: Dissolve 60.5 g Tris Base in 400 ml ddH2O in measuring glass cylinder (stir bar, magnetic stirrer), and adjust the pH to 6.8 with HCl and fill up with ddH2O to a final volume of 500 ml
Ø 3M Tris-HCl pH 8.8: Dissolve 181.5 g Tris Base in 400 ml ddH2O in measuring glass cylinder (stir bar, magnetic stirrer), and adjust the pH to 8.8 with HCl and fill up with ddH2O to a final volume of 500 ml
Ø 10 % SDS (sodium dodecyl sulfate, Biorad No. 161-0302): Dissolve 10 g of SDS in 90 ml of ddH2O in a beaker with a stir bar on a magnetic stirrer at 68 °C (assist dissolving process). Fill up with ddH2O to a final volume of 100ml. Sterile filtration is not necessary. Do not autoclave. Store at room temperature
Ø TEMED (N, N, N, N-tetramethylethylenediamine; Biorad, 161-0800); store at 4 °C Ø 10 % APS: Dissolve 1 g ammonium persulfate (Biorad, 161-0700) in 10 ml ddH2O and
freeze 1ml aliquots at -20 °C (APS decomposes at room temperature) Ø 10x electrophoresis /running buffer:
0.25 M Tris base (30.3 g Trizma; Sigma T1503) ,1.9 M glycine (144 g, Biorad 161-0718), 1 % (w / v) SDS (10 g) make up to 1 liter of final volume with ddH2O and stir; no pH adjustment (has a pH 8.3 after dilution); store at room temperature; dilute 1:10 with ddH2O for use
Ø Stacking Gel (5 % Acrylamide) reagents: • 30 % acrylamide / 0.8 % bis-acrylamide • 1 M Tris-HCl pH 6.8
• 10% SDS • ddH2O • TEMED
28 Ø Resolving Gel /Separating Gel (12 % Acrylamide) reagents:
• 30 % acrylamide / 0.8 % bis-acrylamide • 3 M Tris-HCl pH 8.8
• 10 % SDS • ddH2O • TEMED
• 10 % APS (freshly thawed)
1. Assemble glass plates and spacer plates in gel casting apparatus as described in Bio-Rad instruction manual.
2. For a 1.5 mm thick gel prepare a 12 % separating gel solution, by mixing 1.3 ml of 3 M Tris-HCl pH 8.8, with 4 ml acrylamide/bis solution, 0.1 ml of 10 % SDS, 4.6 ml ddH2O, 50 μl ammonium persulfate (APS) solution, and 5 μl TEMED on a magnetic stirrer with a magnetic stir bar.
3. TEMED and ammonium persulfate (APS) should be added at last because once TEMED and APS are added then the solution will polymerize within few minutes. Pour the separating gel solution between glass frames that leave the space for a stacking gel. 4. Fill approximately 0.5 cm height of ddH2O on top of the stacking gel solution before it
polymerizes to produce a smooth, completely level surface on top of the separating gel. 5. Allow separating gel to polymerize for about 45 - 60 minutes. After polymerization pour
off ddH2O.
6. Prepare the stacking gel solution by mixing 1.3 ml of 1 M Tris-HCl pH 6.8 with 1.7 ml acrylamide/bis solution, 0.1 ml of 10 % SDS, 7.2 ml ddH2O, 50 μl ammonium persulfate solution, and 10 μl TEMED on the magnetic stirrer with a magnetic stir bar.
7. Pour the stacking gel solution on the top of the polymerized separating gel. Insert comb gently before the stacking gel solution starts to polymerize. The plastic comb in the polymerizing stacking gel provides the opening of the wells for the sample application. Allow stacking gel to polymerize for 45 - 60 minutes.
Electrophoresis
1. Once the stacking gel has polymerized, carefully remove gel cassette from the casting stand and place it onto the electrode assembly (with the short plate facing inward). Place the second gel cassette on the other side of the electrode assembly.
2. Fill the electrode assembly (inner chamber) with 1 x electrophoresis buffer to just under the edge of spacer plate and carefully remove the plastic comb from the gel cassette.
29 3. Load 10 μl of prestained molecular weight marker in one of the wells and then 25 μl of
each sample into the wells with a pipet using gel loading tips.
4. Carefully place the electrode assembly with loaded samples into clamping frame and both will put into the Mini-PROTEAN Tetra tank and fill the tank (outer chamber) with 1 x electrophoresis buffer.
5. Connect the red and black electrical leads into the matching colored electrodes of the electrode assembly.
6. Run the gel either at constant voltage 200 V till bromophenol dye has migrated to the bottom of the gel. Normally it takes 40 - 50 minutes.
7. When the dye has reached the bottom of the gel remove the electrode assembly.
Western blot analysis
The western blot serves as an immunochemical detection method for the recombinant protein GFP in infected cells. The expression rate should be quantified over time and displayed in a diagram. For this purpose, the proteins from all samples are separated by SDS-PAGE are transferred by means of a semi-dry blotter onto a PVDF membrane and the recombinant GFP is visualized by a sequential incubation with primary and fluorescence-coupled secondary antibodies. Detection is achieved by using a fluorescence imager.
Material list:
Ø 10 x Transfer Buffer:
• Dissolve 200 mM Tris base (24.4 g/l) and 1.5 M (112.6 g/l) glycine in 900 ml ddH2O in a measuring cylinder and fill with ddH20 up to a final volume of 1L Ø 1 x Blotting Buffer:
• 100 ml 10x transfer buffer • 200 ml ethanol
• 700 ml ddH2O. Chill the buffer at 4 °C Ø Methanol
Ø PVDF (polyvinylidene difluoride) membrane, Hybond-LFP (Amersham) from GE Healthcare
Ø 3-meter blotting paper Ø Semi-dry blotter
1. After the gel electrophoretic separation of the proteins, the stacking gel is removed with a rollerblade. Stored the gel temporarily in blotting buffer to prevent dehydration. However, if it stays too long in the blotting buffer, the gel swells (change in gel size), and the protein bands become broader.
30 2. Cut a piece of PVDF membrane to the size of the gel or larger and handle it only with flat forceps. Wet the membrane for a short time in methanol and then immerse it in a tray filled with blotting buffer.
3. Cut the blotting paper also in six gel sized pieces. Soak all pieces by placing in a tray filled with blotting buffer.
4. Wet the anode and cathode plate with a very small amount of blotting buffer.
5. Place three layers of blotting paper onto the anode plate. Roll a pipet or test tube over the surface of the paper to exclude air bubbles.
6. Place the membrane on to the blotting paper set and remove the bubbles.
7. Carefully place the gel on top of the membrane, aligning the stack as perfect as possible and place another three layers of blotting paper on it.
8. Carefully place the cathode plate onto the stack and run the transfer at either 25 V or less than 3 mA/cm2 current for 30 minutes.
9. After the transfer, carefully remove the blotting paper with tweezers. Mark the membrane with a ballpoint pen to remember at which side gel was transferred.
10. Remove the gel and place the membrane in a dish with PBS- and wash it briefly to wash off any gel residue. The membrane can be used directly for immunodetection or can be stored in some PBS- covered foil at 4 °C.
Immunodetection Material list:
Ø Chemiluminescence / Fluorescence Imager Fusion Xpress from Peqlab Ø Washing buffer: dissolve 0.1 % Tween 20 (0.5 g) in 500 ml 1x PBS
-Ø Blocking solution: dissolve 5 % Milk powder (5g) in 100 ml washing buffer
Antibody Name Dilution factor (in
blocking solution)
Primary antibody Mouse anti-His 1: 2000
Secondary antibody IRdye 800CW anti mouse 1: 5000
31 1. Incubate the membrane in 50 ml blocking buffer for one hour on a rocking table at room
temperature to prevent non-specific binding of the antibody.
2. Discard the blocking buffer and incubate the membrane with 1:2000 dilution of primary antibody in blocking buffer in an enclosed plastic bag (sealed) for one hour at room temperature on a rocking table.
3. Remove the primary antibody and wash the membrane in the tray three times for five minutes with 50 ml washing buffer.
4. Discard the washing buffer and incubate the membrane with 1:5000 dilution of secondary antibody in blocking buffer in an enclosed plastic bag (sealed) for one hour at room temperature on a rocking table.
5. Remove the secondary antibody and wash the membrane in the tray three times for five minutes with 50 ml washing buffer.
6. Discard the washing buffer and wash the membrane for five minutes in the tray with 50 ml 1 x PBS-.
7. Dry the membrane on a blotting paper to reduce the background signal and analyse membrane for an exposure time of 50 seconds in the dark room with the infrared fluorescence imager ("Fusion Express", 5.5 million-pixel CCD camera with a motorized zoom lens, motorized filter wheel and cooling system, darkroom, control, and analysis software).
32
3 Results
The first aim of this master thesis was to efficiently amplify a baculovirus stock recombinant for GFP using as little virus stock as possible. The second task was to optimize the expression of a recombinant protein by infecting Sf9 cells with the amplified virus in spinner flasks. In order to estimate the virus titers and to analyse the protein expression, signals from the Green Fluorescence Protein (GFP) were followed. An experimental outline of the entire process and time scale required for each step are shown below in figure 3.1 and 3.2.
Figure 3.1: Schematic representation of the experimental steps involved in optimal baculovirus amplification. (A) amplification of baculovirus by infecting Sf9 cells in T-25 flasks for five days and infection of amplified virus for two days in 24-well plate to determine the titer (B) titer estimation of amplified baculovirus using fluorescence microscopy analysis.
A
33 Figure 3.2: The experimental outline for optimal recombinant protein expression. (A) expanding Sf9 cells in spinner flasks and (B, C) determining protein expression by infecting Sf9 cells in spinner flasks without protease inhibitor (B) or in the presence of protease inhibitor (C) for up to five days. Samples of cells were taken every day and analysed for GFP expression by western blotting. [Abbreviations: BV (Baculovirus), FCS (Fetal Calf Serum), PI (Protease Inhibitor cocktail), DMSO (Dimethyl Sulfoxide)].
C B A
34
3.1 Infection of Sf9 cells with baculovirus
The first experimental goal was to amplify the baculovirus stock as described in figure 3.1. For this virus amplification step, baculovirus stock of the previous generation (P3) containing recombinant GFP-DNA was used to infect Sf9 cells in T-25 flasks. The aim was to achieve maximal virus amplification using only little amount of virus stock. For this purpose, Sf9 cells grown in T-25 flasks were infected with 20 μl and 40 μl of P3 recombinant baculovirus. After that, flasks were stored in an incubator at 27 °C for 120 hours. Non-infected Sf9 cells were evenly distributed and had a typical round shape. These cells were firmly adherent to the bottom surface of the flask, as shown in (see an arrow in figure 3.3 A). Upon infection, cells start to increase in size, due to the intracellular accumulation of the produced virus, and they begin to detach due to their
decreased viability (see an arrow in figure 3.3 B). These morphological changes were an indication
of successful infection, observed using an inverted light microscope with 100 x magnification.
Non-infected Sf9 cells Bavulovirus-infected Sf9 cells
Figure 3.3: Cell images showing the difference between non-infected and baculovirus-infected Sf9 cells grown in T-25 flasks. Image A represents non-infected subconfluent Sf9 cells, which are similar in size, well attached to the bottom surface and exhibiting circular morphology. Image B represents Sf9 cells, infected with 40 μl of P3 recombinant baculovirus, 120 hours post-infection. Their shape is irregular, and they appear larger in their size as shown with an arrow. These larger cells would later burst open and release the virus into the medium. Both images were taken using an inverted light microscope with 100 X magnification. The shown scale bar is 100 μm for both images.
35
3.2 Analysis of infection efficiency and titer of the amplified
baculovirus using immunofluorescence microscopy
After infection for 120 hours, the collected amplified baculovirus stocks were separated from detached cells by a using centrifuge at the centrifugal force of 500 x g for 5 minutes. Amplified virus stocks generated using 20 μl and 40 μl of P3 recombinant virus were termed as P4V1 and P4V2, respectively.
3.2.1 Fluorescence microscopic analysis of Sf9 cells infected with the amplified
baculovirus stocks
To investigate the infection efficiency of the two amplified virus stocks, Sf9 cells were seeded into 24-well plates on glass coverslips for 24 hours and infected with 10 μl, 25 μl and 50 μl of P4V1 and P4V2 each (see fig. 3.1A). 48 hours post-infection, the cells were fixed using paraformaldehyde (PFA) and analysed under the fluorescence microscope. The number of cells infected with recombinant baculovirus containing the gene of GFP was followed by its green fluorescence in order to determine the infection efficiency. During the fluorescence microscope analysis, nuclei of the Sf9 cells grown on the coverslip were observed with the DAPI filter (DNA stain) while infected cells were observed using the FITC filter (GFP). Then, images of the same area were captured using both filters and the overlay of both images was created using Adobe Photoshop software. Images of cells grown on glass coverslips and infected with different amounts (10 μl, 25 μl and 50 μl) of P4V1 and P4V2 are shown in figure 3.4 and 3.5, respectively. It can be observed in both figures that higher amounts of virus stocks used for infection produce higher number of green fluorescence signals from expressed GFP protein for both virus stocks. Next, the infection efficiencies of both virus stocks were calculated to determine the optimal virus stock with the highest virus concentration. To calculate the infection efficiency of P4V1 and P4V2, the total number of cells in the DAPI filtered image (blue) and infected cells of the same area of the coverslip from the FITC filtered image (green) were counted. However, in this work, only 25 % of the area (as shown in Figure 3.4 and 3.5 with red-colored square) of each image was considered for the infection efficiency calculations.
After counting the number of total and infected cells from each image, the infection efficiencies of 10 μl, 25 μl and 50 μl of P4V1 and P4V2 virus stocks are shown in figure 3.6. It is clear from this calculation that for virus stock P4V1, when 10 μl of the virus was used for infection, 5.0 % of the cell population was infected. When the volume of P4V1 virus was increased by 2.5 and 5 times, 10.1 % and 17.4 % of the cell population was infected, respectively. Therefore, the increase of the P4V1 dose increases the infection efficiency but not in the same proportion as that of the virus dose i.e., 2.5 and 5 times of virus volume increase produced only 2 and 3.5 times of increase in infection efficiency. Similarly, infection efficiency of P4V2 for volumes of 10 μl, 25 μl and 50 μl was 11.8 %, 23.5 % and 47.9 %, respectively. That means, when the dose of P4v2 was increased by 2.5 and 5 times, the infection efficiency increased by 2.2 and 4.5 times, respectively. So, there is a closer
