3.3 Baculoviral
Expression of hKLK7
in Hi5 Cells
The protocol describes the expression and purification of the
secreted KLK7 in Hi5 cells in 500 mL scale using the BEVS
technology (see Note 9).
1. [Day 3, Friday] Prepare a 250 mL preculture starting with
0.3–0.4 Â 10
6 Hi5 c/mL. Incubate the culture for 72 h at
27
C and 110 rpm (see Note 5).
2. [Day 0, e.g., Monday] Count the cells of the preparatory culture
and prepare a 500 mL culture containing 1.0–1.5 Â 10
6 Hi5
c/mL by diluting the required volume of the cell suspension
into fresh EX-CELL 405 medium. Infect the cells at a MOI of
0.5–2.0 with the baculoviral stock solution (see Note 10). Mix
gently and incubate the culture at 27
C and 110 rpm for 72 h.
3. [Day 1–3, Tuesday to Thursday] Take samples daily, monitor
cell numbers, viability, and cell diameter in the CASY Cell
Counter to determine the progress of the baculoviral infection.
If cells reach a density above 3 Â 10
6 c/mL, adjust the concentration back to 1 Â 10
6 c/mL by adding fresh EX-CELL
405 culture medium.
4. [Day 3, Thursday] Harvest secreted target proteins 48 h after
total proliferation stop by centrifuging the cell suspension for
15 min at 1000 Â g and store the supernatant at 4
C after
sterile filtration (0.45 μm) until purification. Prevent microbial
growth by adding 0.05% sodium azide.
3.4 Preparation
of the Baculoviral
Stock Solution
1. [Day 3, Friday] Prepare a 150 mL Sf21 preculture starting with
0.3–0.4 Â 10
6 c/mL in EX-CELL 420 medium. Incubate the
culture for 72 h at 27
C and 110 rpm (see Note 5).
2. [Day 0, e.g., Monday] Count the cells of the preparatory culture
and prepare a 500 mL culture containing 0.5 Â 10
6 Hi5 c/mL
by diluting the required volume of the cell suspension into
fresh EX-CELL 420 medium. Infect the cells with a MOI of
0.2 with the baculoviral transfection supernatant or use 5% v/v
of a nontitered viral stock. Mix gently and incubate the culture
at 27
C and 110 rpm for 4 days.
3. [Day 1–4, Tuesday to Friday] Monitor the cell number, viability, and the cell diameter (CASY cell counter). The cells should
be split to 1 Â 10
6 c/mL by adding fresh EX-CELL
420 medium once they reach 2 Â 10
6 c/mL.
4. [Day 4, e.g., Friday] The viral supernatant was harvested
48 hours after an increase in cell diameter of 3 μm was
observed.
3.5 Protein
Purification by Affinity
Chromatography
Purification and maintenance of the columns and chromatography
systems were done according to the protocols recommended by the
manufacturing company.
Expression of Recombinant Antibodies in Different Formats
69
Expression of hKLK7
in Hi5 Cells
The protocol describes the expression and purification of the
secreted KLK7 in Hi5 cells in 500 mL scale using the BEVS
technology (see Note 9).
1. [Day 3, Friday] Prepare a 250 mL preculture starting with
0.3–0.4 Â 10
6 Hi5 c/mL. Incubate the culture for 72 h at
27
C and 110 rpm (see Note 5).
2. [Day 0, e.g., Monday] Count the cells of the preparatory culture
and prepare a 500 mL culture containing 1.0–1.5 Â 10
6 Hi5
c/mL by diluting the required volume of the cell suspension
into fresh EX-CELL 405 medium. Infect the cells at a MOI of
0.5–2.0 with the baculoviral stock solution (see Note 10). Mix
gently and incubate the culture at 27
C and 110 rpm for 72 h.
3. [Day 1–3, Tuesday to Thursday] Take samples daily, monitor
cell numbers, viability, and cell diameter in the CASY Cell
Counter to determine the progress of the baculoviral infection.
If cells reach a density above 3 Â 10
6 c/mL, adjust the concentration back to 1 Â 10
6 c/mL by adding fresh EX-CELL
405 culture medium.
4. [Day 3, Thursday] Harvest secreted target proteins 48 h after
total proliferation stop by centrifuging the cell suspension for
15 min at 1000 Â g and store the supernatant at 4
C after
sterile filtration (0.45 μm) until purification. Prevent microbial
growth by adding 0.05% sodium azide.
3.4 Preparation
of the Baculoviral
Stock Solution
1. [Day 3, Friday] Prepare a 150 mL Sf21 preculture starting with
0.3–0.4 Â 10
6 c/mL in EX-CELL 420 medium. Incubate the
culture for 72 h at 27
C and 110 rpm (see Note 5).
2. [Day 0, e.g., Monday] Count the cells of the preparatory culture
and prepare a 500 mL culture containing 0.5 Â 10
6 Hi5 c/mL
by diluting the required volume of the cell suspension into
fresh EX-CELL 420 medium. Infect the cells with a MOI of
0.2 with the baculoviral transfection supernatant or use 5% v/v
of a nontitered viral stock. Mix gently and incubate the culture
at 27
C and 110 rpm for 4 days.
3. [Day 1–4, Tuesday to Friday] Monitor the cell number, viability, and the cell diameter (CASY cell counter). The cells should
be split to 1 Â 10
6 c/mL by adding fresh EX-CELL
420 medium once they reach 2 Â 10
6 c/mL.
4. [Day 4, e.g., Friday] The viral supernatant was harvested
48 hours after an increase in cell diameter of 3 μm was
observed.
3.5 Protein
Purification by Affinity
Chromatography
Purification and maintenance of the columns and chromatography
systems were done according to the protocols recommended by the
manufacturing company.
Expression of Recombinant Antibodies in Different Formats
69