Protein expression using the baculovirus expression system

Over the past decade the use of baculoviruses has become an important tool for overexpressing recombinant proteins in eukaryotic cells 149,150. Unlike the bacterial expression systems, this eukaryotic system uses most of the protein modification, processing, folding and transport machinery that is present also in higher eukaryotes. Thus, with this system, most of the over-expressed proteins exhibit proper biological activity and function 151_.

For the purpose of this thesis, the expression of the different protein constructs was carried out with the Bac-to-Bac® baculovirus expression system (Invitrogen) in the Sf9 insect cell line (Section 4.1.5). One of two major components of the Bac-to-Bac®_{-system is the vector into which the gene or genes of interest is/are}

cloned. In this thesis, vectors containing one or two promoters, and thus one or two expression cassettes, were used. Dual vectors, i.e., vectors that carry two such expression cassettes, allow for the expression of two different proteins that are encoded by two different genes while cloned into the same plasmid. Both, the polyhedrin (pPH) and the p10 promoter in the wildtype baculovirus AcMNPV promote transcription of two major late viral proteins. Both, the polyhedrin and p10 gene were replaced by a foreign gene in the here used recombinant baculoviruses. Thereby recombinant protein expresssion under the control of those two promoters was made possible.

The second major component of the Bac-to-Bac® system is represented by an E. coli strain (DH10Bac Competent E. coli) that is used as host for the before

generated plasmid, consisting of transfer vector and gene(s) of interest. DH10Bac Competent E. coli cells contain a baculovirus shuttle vector, referred

to as bacmid. After transformation into these cells, transposition between transposons of the vector and the bacmid occurs. This recombinant bacmid is the basis to then generate the recombinant baculovirus.

An overview of the key steps for baculovirus protein expression is given in Figure 13.

4.3.3.1 Construction of the recombinant transfer vector for protein

expression

In this thesis two dual vectors and one mono vector were used to generate recombinant transfer vectors for the expression of the herein used protein constructs (vector maps are provided in Section 7). The two dual vectors, named hereafter p2bac/pfastbac and pFastBacDual, mainly differ in their multiple

cloning sites downstream of the p10 and polyH promoter. The presence of these two strong promoters allows for the simultaneous expression of two different proteins encoded on the same plasmid. The expression cassette is flanked by the left (L) and right (R) arms of Tn7, which are mini Tn7 elements that permit site- specific transposition of the gene of interest into the baculovirus genome 152. It also contains a SV40 polyadenylation signal, which permits efficient transcription termination and polydenylation of mRNA 153. In addition the vectors contain an ampicillin and gentamicin resistance gene, where the first one allows selection of the plasmid in E. coli XL 1-Blue cells, while the latter one permits selection of the recombinant bacmid in E. coli DH10Baccells.

Except for the fact that the mono vector pFastBac1 possesses the polyH

promoter only, all the genes and regions that were described for both the dual vectors are present also in the pFastBac1 vector (vector maps are provided in

Figure 13. Protein expression via the Baculovirus Bac-to-Bac® system − An overview.

The flowchart illustrates the required steps for the successful protein expression via the Bac- to-Bac® system.

4.3.3.2 Generation of the recombinant bacmid

Once the recombinant vector was generated, for transposition into the baculovirus shuttle vector (bacmid) the amplified and purified DNA was transformed into DH10Bac E. coli cells. This strain of E. coli cells contains the bacmid with a

mini-attTn7 attachment site, which is inserted in a segment of DNA encoding the LacZα peptide, and a helper plasmid. Generation of the recombinant bacmid is

achieved via site-specific transposition between the mini-Tn7 element of the donor vector and the mini-attTn7 attachment site on the bacmid (Figure 14).

For transformation, chemically competent DH10Bac E. coli cells (aliquots of 50 µl) were thawed on ice and incubated with 10 ng DNA (in 5 µl TE buffer) 30 minutes on ice. The heat shock was performed for 45 seconds at 42°C, followed by immediate incubation on ice. Subsequently, 900 µl of S.O.C. medium was added and the suspension was incubated in a shaker device at 225 rpm, for 4 hours at 37°C.The Tn7 transposition functions are provided by a helper plasmid, which encodes for the transposase and confers resistance to tetracycline. Thus, successful recombination leads to the disruption of the LacZ gene on the bacmid, which in turn allows for blue-white screening in the presence of the chromogenic substrate Bluo-gal and the inducer IPTG.

Figure 14. Generation of recombinant bacmid by site-specific transposition.

Left: before transformation of transfer vector into DH10Bac cells; middle: transposition of the

gene of interest and the gene encoding for gentamicin resistance into the bacmid (black arrows); right: recombinant bacmid with Tn7-element of transfer vector. LacZ gene and genes encoding resistance for indicated antibiotics are color-coded.

(50 µg/ml) kanamycin, (7 µg/ml) gentamicin and (10 µg/ml) tetracycline. The plates were incubated for 48 hours at 37°C to grow single colonies. Two to ten single large and white colonies were picked and re-streaked to confirm the white (i.e., recombined bacmid) phenotype on new LB plates supplemented with same concentrations of kanamycin, gentamicin, tetracycline, Bluo-Gal and IPTG.

4.3.3.3 Isolation of the recombinant bacmid DNA

Single large and white colonies from the re-streak plates were picked and grown to saturation overnight in culturing tubes containing 6 ml LB medium supplemented with kanamycin, gentamicin, tetracycline at the same respective concentrations as the ones applied before. Cells were pelleted at in a Rotanta 460R swinging bucket centrifuge (Hettich) at 2,500 x g for 15 minutes at 4°C (Rotanta 460R swinging bucket centrifuge, Hettich).

Plasmid isolation and purification was performed with buffers P1, P2 and P3 from the Qiagen® Midiprep kit, using the following protocol, while all centrifugation steps were carried out at 16,000 × g for 1 minute at room temperature in an

Eppifuge.

Pelleted cells were resuspended in 500 µl of P1 buffer and then lysed by adding 500 µl P2 buffer, followed by incubation for 5 minutes at room temperature. 500 µl of P3 buffer was added and incubated for additional 10 minutes on ice. The solution was cleared by centrifugation for 15 minutes. The supernatant containing the plasmid DNA was briefly mixed with 1.6 ml ice-cold isopropanol and incubated for 30 minutes on ice. The precipitated DNA was pelleted by centrifugation for 15 minutes, followed by a wash step with 1 ml of ethanol (70%) before again pelleted by centrifugation for 5 minutes. The pellets were then air-dried for 15 minutes and subsequently dissolved in 40 to 50 µl of double-distilled water. Before storage at - 20°C, the DNA concentration of all purified plasmids was determined (Section 4.2.2.7).

The insertion of the gene of interest was confirmed by PCR using promoter- and gene-specific primers. The correct orientation of the transposon inserted into the bacmid was checked by combining a (gene-) internal forward and an external reverse primer.

4.3.3.4 Transfection of Sf9 insect cells with recombinant bacmid

For transfection it is crucial that the bacmid DNA is clean and free from any salts, as contaminants will kill the cells and salt will interfere with lipid complexing, eventually leading to low levels in transfection efficiency (Invitrogen, 2004).

For transfection, per probe 2 ml of freshly diluted Sf9 cells at a density of 0.5*106 cells/ml were seeded into one well of a 6-well tissue culture plate. The cells were incubated light protected for 15 to 20 minutes at 28°C, allowing the cells to attach to the surface of the tissue culture plate.

In the following, 1 to 10 µl of recombinant bacmid DNA solution was mixed with 5

µl of the cationic lipid Cellfectin® reagent (Invitrogen) into 200 µl Sf-900 II SFM medium (no additives). Cellfectin® _{is a 1:1.5 (m/m) liposome formulation of the}

cationic lipid N, NI_{, N}II_{, N}III_{-tetramethyl-N, N}I_{, N}II_{, N}III_{-tetrapalmitylspermine (TM-}

TPS) and dioleoyl phosphatidylethanolamine (DOPE), which coats the DNA and thereby facilitates transfection (Figure 15).

The mixture was incubated for 15 minutes at 28°C before 1 ml Sf-900 II SFM medium was added. As a next step, the medium from the seeded cells was removed and the cells were washed twice with 2 ml Sf-900 II SFM medium (no additives). Subsequently, the cells were overlaid with the Cellfectin®-DNA

Figure 15. Preparing the bacmid for transfection into insect cells via Cellfectin® reagent.

The cationic lipid (Cellfectin® reagent) coats the bacmid DNA for the subsequent chemical transfection into insect cells.

Then the transfection mix was removed and supplemented with 3 ml of fresh Sf- 900 II SFM medium (containing 10% serum and 1% gentamicin, 10 mg/ml), followed by further incubation for two to three days at 28°C.

Virus-infected insect cells are characterized by increased cell size. In addition, after transfection the formerly surface-adhered cells detach from the plate they were seeded on and eventually undergo cell lysis, resulting in the release of virus into the medium (Figure 16).

48 to 60 hours after transfection, the initial virus (V0 generation) was harvested,

sterile-filtered using a 0.22 µm syringe filter and stored light-protected at 4°C.

4.3.3.5 Amplification of baculovirus V

generation

The V0 viral stock is a small-scale, low-titer stock and is used to infect a larger

volume of yet uninfected cells to generate a high-titer V1 (or higher) virus

generation. For viral amplifications always cells from suspension culture were used. For an efficient amplification, prior infection the viability of the cell stock was

Figure 16. Schematic showing the time course from cell transfection until harvesting of released virus.

Top left: Cellfectin-mediated transfection of insect cells with bacmid DNA; top right: following transfection, within approx. 48 to 60 hours cells produce viral proteins, that eventually assemble into intact lytic viral particles; free virus is eventually harvested and used for further virus amplification.

desired. Typically, 100 ml of uninfected cells were infected with 100 to 300 µl of V0 virus. Amplifications were incubated shaking at 110 rpm and 28°C. To

determine the progress of the individual amplification cell viability and density was determined daily. As long as the cell density was higher than 0.5*106 cells/ml, the cell suspension was diluted to 0.5*106 cells/ml, while keeping the volume constant. Virus was harvested when the cell density stayed below 0.5*106 _cells/ml

for additional 48 hours. The harvested V1+x generations can be stored light

protected at 4°C up to several months.

4.3.3.6 Protein expression in Sf9 insect cells

For all proteins over-expressed via the baculovirus expression system, expression was carried out in insect Sf9 cells from suspension culture.

300 ml to 800 ml freshly diluted Sf9 cells at a density of 2*106 _{cells/ml (viability}

greater than 95%) were used for protein expression from suspension culture in 1 to 6 L-glass spinner flasks. Typically, 2 to 5% vol. of recombinant virus encoding for the gene(s) of interest, were used to transfect the cells. Depending on the protein to be expressed, the suspension of transfected cells was incubated for 45 to 80 hours in a shaker device at 110 rpm and 28°C.