8. [Day 1–7, e.g., Tuesday to Monday] Take samples daily and
determine transfection efficiency in the flow cytometer
(Guava EasyCyte) or/and determine target protein expression
by a suitable technique (SDS-PAGE, slot blot, or
Western blot).
9. [Day 2, e.g., Wednesday] Add 250 mL fresh F17 to the cells as
well as 12.5 mL of the TN1-Stock (20%) ¼ 0.5%.
10. [Day 3, e.g., Thursday] Add 7.5 mL of the glucose stock
(300 g/L) ¼ 4.5 g/L.
11. [Day 4, e.g., Friday] Add 25 mL of the valproic acid stock
(75 mM) ¼ 3.75 mmol/L.
12. [Day 7, e.g., Monday] Harvest secreted target proteins by
centrifuging the cell suspension for 15 min at 1000 Â g and
store the supernatant at 4
C until purification. Depending on
the protein, it might be required to add protease inhibitor. For
intracellular proteins, snap freeze the cell pellet and store at
À80
C until cell lysis and purification.
The cloned expression vectors were transfected single or in
pairs into HEK293-6E cells, and the culture was grown for
168 h. The viability of the cells remained >80%, and the efficiency
of transfection varied from 20% to 80% for the different experiments (see Fig. 3). All the supernatants contained sufficient antibody for subsequent purification of the protein samples. The
quality of the purified antibodies was validated by SDS-PAGE.
Total volumetric yield and specific cellular yield of the recombinant
antibodies are presented in Fig. 4.
3.2 Transient Protein
Expression in Hi5 Cells
The protocol is described in Karste et al. MiMB [1]. Here, we show
the expression and purification of the secreted KLK7 in Hi5 cells in
500 mL scale. The employed vectors contain the optimal expression cassette with the strong OpIE2 promoter (see Note 4, [20]).
1. [Day 1, e.g., Friday] Prepare a 150 mL preculture starting at
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 100 mL culture containing 5 Â 10
6 Hi5 c/mL by
centrifuging the required volume of the cell suspension at
180 Â g for 4 min. Discard the supernatant and resolve the
cell pellet in 100 mL fresh EX-CELL 405.
3. Incubate the culture at 27
C and 110 rpm for 1 h.
4. Mix 425 μg of the expression plasmid and 25 μg of the
control plasmid, e.g., pOpIE2-eGFP-HA, coding for eGFP
(see Note 6).
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Jens Ko ¨ nig et al.
determine transfection efficiency in the flow cytometer
(Guava EasyCyte) or/and determine target protein expression
by a suitable technique (SDS-PAGE, slot blot, or
Western blot).
9. [Day 2, e.g., Wednesday] Add 250 mL fresh F17 to the cells as
well as 12.5 mL of the TN1-Stock (20%) ¼ 0.5%.
10. [Day 3, e.g., Thursday] Add 7.5 mL of the glucose stock
(300 g/L) ¼ 4.5 g/L.
11. [Day 4, e.g., Friday] Add 25 mL of the valproic acid stock
(75 mM) ¼ 3.75 mmol/L.
12. [Day 7, e.g., Monday] Harvest secreted target proteins by
centrifuging the cell suspension for 15 min at 1000 Â g and
store the supernatant at 4
C until purification. Depending on
the protein, it might be required to add protease inhibitor. For
intracellular proteins, snap freeze the cell pellet and store at
À80
C until cell lysis and purification.
The cloned expression vectors were transfected single or in
pairs into HEK293-6E cells, and the culture was grown for
168 h. The viability of the cells remained >80%, and the efficiency
of transfection varied from 20% to 80% for the different experiments (see Fig. 3). All the supernatants contained sufficient antibody for subsequent purification of the protein samples. The
quality of the purified antibodies was validated by SDS-PAGE.
Total volumetric yield and specific cellular yield of the recombinant
antibodies are presented in Fig. 4.
3.2 Transient Protein
Expression in Hi5 Cells
The protocol is described in Karste et al. MiMB [1]. Here, we show
the expression and purification of the secreted KLK7 in Hi5 cells in
500 mL scale. The employed vectors contain the optimal expression cassette with the strong OpIE2 promoter (see Note 4, [20]).
1. [Day 1, e.g., Friday] Prepare a 150 mL preculture starting at
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 100 mL culture containing 5 Â 10
6 Hi5 c/mL by
centrifuging the required volume of the cell suspension at
180 Â g for 4 min. Discard the supernatant and resolve the
cell pellet in 100 mL fresh EX-CELL 405.
3. Incubate the culture at 27
C and 110 rpm for 1 h.
4. Mix 425 μg of the expression plasmid and 25 μg of the
control plasmid, e.g., pOpIE2-eGFP-HA, coding for eGFP
(see Note 6).
66
Jens Ko ¨ nig et al.