Entrevista

A 50 μl vial of competent TOP10 cells was defrosted on ice. 5 μl of the ligated construct

was added and the cells incubated on ice for 30 mins. The cells were given a heat shock in a water bath at 42◦C for 30 seconds and then returned to ice for 2 mins. 250 μl of S.O.C

media was added and then the cells shaken at 225 rpm, 37◦C for 1 hour.

Agarose plates containing 25 µg/ml zeocin (Section 2.1.16) were prepared and pre-warmed to 37◦C. 5–200 μl of the transformation reaction was spread onto the agarose

Restriction digest analysis of DNA from positive colonies

Single colonies were grown overnight at 37◦C in 5 ml cultures with vigorous shaking (225 rpm) and DNA purified by mini-prep (Section 2.2.11). DNA was then digested using restriction enzymes NcoI and BglII with NEB buffer 3.1 for the pFuse-3.4 construct, and NcoI and EcoRI with NEB buffer 3.1 for the pFuse-3.8 construct.

The digestion reaction consisted of the following solution:

• Restriction enzyme 1 1μl • Restriction enzyme 2 1μl • Compatible Buffer 5 μl • BSA (10x) 5μl • pFUSE-DBL construct 1μg • dH₂O up to 50 μl.

Restriction digests were incubated at 37◦C for 2 hours and then the product visualised by gel electrophoresis (Section 2.2.5). DNA preparations with the correct inserts were identified and sent for DNA sequencing to confirm (Section 2.2.13).

Maxi-prep

1 μl DNA containing the confirmed pFUSE-DBL constructs was transformed directly

into TOP10 cells and colonies picked and grown for 8 hours at 37◦C (225 rpm) in 5 ml cultures before being transferred to 250 ml cultures and grown overnight under the same conditions. The DNA was then extracted using QIAprep Spin Maxiprep kit (Section 2.2.14) and sequenced (Section 2.2.13).

Primers for sequencing of the pFUSE constructs

Primers for the sequencing of the pFUSE constructs were designed using Primer3Plus http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ (Table 2.2).

3.3.8 Expression of 3.4 and 3.8 DBL recombinant protein

Once the DBL-Fc fusion constructs had been made they were expressed in mammalian cells.

CHO-KI cell transfection

CHO-KI cells were prepared for transfection (Section 2.3.1) by growing cultures until the cells were 50−80% confluent. Cells were transfected using the FuGENE 6 Transfection Reagent. In a sterile tube containing 3μg of plasmid DNA, 100μl of medium was added

and 9 μl of FuGENE 6 was added directly to the mixture. The tube was vortexed gently

to mix and incubated at room temperature for 15 mins.

5 x 105cells were added to 10 ml DMEM and, following incubation, the DNA/FuGENE was added to the CHO-KI cells, mixed and added to tissue-culture plates. 24 hours after transfection, the medium was replaced with DMEM selection media (Section 2.1.15).

Production of polyclonal population

After 7 - 9 days, transfected cells were passaged to expand the polyclonal population. Cells were washed in 10 ml sterile PBS before coating with trypsin and incubating for 5 mins at 37◦C. Cells were resuspended in DMEM selection medium and expanded in small then medium-sized flasks. Polyclonal populations were frozen down for long-term storage when 80% confluent (Section 2.3.5).

Production of monoclonal population

Polyclonal populations were diluted to 10 cells/ml and 100μl plated in each well of 96 well

plate. 10 μl of supernatant from wells where single cells could be seen to have formed a

colony was harvested after 9 days, and protein detected by immunoblot analysis (Section 2.4.2). The colonies found to be producing the most protein were expanded further. A single colony was transferred to a single well of a 24-well plate before being expanded to a small flask, and subsequently into medium flasks to expand the clonal cell line. Finally, clonal cell lines were cultured in 4 large tissue-culture flasks for 10 days to produce 200 ml supernatant containing protein from the monoclonal population. Timescales for expanding the single colony up to medium flasks was flexible depending on the rate of cell growth. However, all cultures seeded into large flasks were cultured for a period of exactly 10 days

to produce protein.

FPLC purification of recombinant DBL protein

Cell supernatants containing protein were filter sterilised by vacuum using Steritop GP Sterlisation Unit 0.22 μm PES membrane (Millipore). Sterile cell supernatants were

purified using the liquid chromatography system ¨AKTA-FPLC Frac-950 (Amersham Biosciences/GE Healthcare, Uppsala, Sweden). A 1 ml protein-G sepharose HiTrap column (GE Healthcare) was used and equilibrated by washing with a minimum 20 ml of HBSS buffer. Supernatant was loaded onto the column at a flow rate <0.5 ml/min. Once fully loaded, the column was thoroughly washed with a minimum 30 ml of HBSS buffer to remove any unbound sample. Protein was eluted using an increasing gradient of 0.1M glycine-HCl, pH 2.5 and collected in 2 ml fractions. Eluates were neutralised with 1M Tris-HCl pH 9 and stored with 0.2% sodium azide at 4◦C.

Immunoblot to test for the presence of recombinant DBL protein

FPLC fractions were tested for the presence of recombinant DBL protein using immunoblot analysis. 5μl of each fraction was blotted onto nitrocellulose and blocked with PBS-tween

plus 5% milk for 2 hours at 37◦C on a rocker. The nitrocellulose was washed in PBST and anti-human IgG (Fc-specific) Alkaline Phosphatase antibody (Sigma A9544) was added at a dilution of 1:5,000 and incubated overnight at 37◦C on a rocker. The blot was developed using NBT/BCIP tablets (Table 2.12) according to manufacturer’s instructions. Fractions containing protein were combined and concentrated by ultrafiltration (Section 2.4.3). The flow-through liquid was also tested for protein; if protein was still present in the flow-through it was passed over the column again in order to extract all the sample.