53. Store at 4
C protected from light. This virus is the P1 virus
stock. P1 is sufficient for initial protein expression studies. If
large volumes of virus are required, amplify P1 to obtain P2.
54. Determine the titer of the P1 virus using the endpoint dilution
assay.
3.2.2 Secretory
Expression Using Sf9 Insect
Cells/Baculoviruses
1. Day 1: Expansion of Sf9 cells should be prepared in advance, so
that a sufficient number of cells is available on this day. To
expand Sf9 cells, determine the total number of cells and
percent viability using a LUNA automated cell counter and
Trypan blue exclusion, and ensure that the density of the cells
is 1.5 Â 10
6 cells/mL. Based on the volume of cells needed
(0.5–2 L), calculate the volume of the medium needed to
dilute the culture. For 0.5 L, 7.5 Â 10
8 cells and ~ 0.4 L of
PSFM-J1/FBS-PS medium will be needed and a 3-L Fernbach
flask (Vent filter cap/Baffle bottom).
2. Add P1 virus at a multiplicity of infection (MOI) of 1 to infect
Sf9 cells at a density of 1.5 Â 10
6 cells/mL (see Note 8).
3. Incubate the cells at 27
C with agitation (120 rpm).
4. Days 2–4: Monitor the mCherry-derived fluorescence in the
culture supernatant over the elapsed time. Take an aliquot of
the Sf9 culture, spin down the cells using a table top centrifuge,
and use 200 μL of the supernatant to measure the fluorescence
(excitation: 587 nm; emission maximum: 610 nm).
5. Day 4: Collect the culture supernatant 96 h after infection by
centrifugation for 15 min at 6000 Â g at 4
C. Discard the cell
pellet. Adjust the pH of the supernatant to ~8.0 by adding
1/20 volume of 1 M Tris-HCl (pH 8.0).
3.2.3 Purification
of Genetically Encoded
Fluorescent Fab Fragments
1. Day 1: (IMAC) Use 20 mL of Ni Sepharose excel resin (40 mL
of 50% slurry) per 1 L of Sf9 culture, pour the slurry into a glass
Econo-Column, and equilibrate with five CVs of buffer A.
2. Add the Sf9 culture supernatant directly to the column, and
collect the flow-through fraction.
3. Wash the column with 10 CVs of buffer A.
4. Elute the polyprotein of interest from the column by adding
three CVs of buffer B and collect the eluate.
5. Measure the protein concentration of the eluate using a NanoDrop spectrophotometer.
6. Concentrate the protein using an Amicon Ultra-15 (MWCO
30 K) centrifugal concentrator to the volume of 5 mL.
7. (SEC) Inject the 5 mL of concentrated protein sample onto a
HiLoad16/60 Superdex200 column pre-equilibrated with
TBS at a flow rate of 1 mL/min.
Production of Recombinant Antibody Fragments via the iRAT system
97
C protected from light. This virus is the P1 virus
stock. P1 is sufficient for initial protein expression studies. If
large volumes of virus are required, amplify P1 to obtain P2.
54. Determine the titer of the P1 virus using the endpoint dilution
assay.
3.2.2 Secretory
Expression Using Sf9 Insect
Cells/Baculoviruses
1. Day 1: Expansion of Sf9 cells should be prepared in advance, so
that a sufficient number of cells is available on this day. To
expand Sf9 cells, determine the total number of cells and
percent viability using a LUNA automated cell counter and
Trypan blue exclusion, and ensure that the density of the cells
is 1.5 Â 10
6 cells/mL. Based on the volume of cells needed
(0.5–2 L), calculate the volume of the medium needed to
dilute the culture. For 0.5 L, 7.5 Â 10
8 cells and ~ 0.4 L of
PSFM-J1/FBS-PS medium will be needed and a 3-L Fernbach
flask (Vent filter cap/Baffle bottom).
2. Add P1 virus at a multiplicity of infection (MOI) of 1 to infect
Sf9 cells at a density of 1.5 Â 10
6 cells/mL (see Note 8).
3. Incubate the cells at 27
C with agitation (120 rpm).
4. Days 2–4: Monitor the mCherry-derived fluorescence in the
culture supernatant over the elapsed time. Take an aliquot of
the Sf9 culture, spin down the cells using a table top centrifuge,
and use 200 μL of the supernatant to measure the fluorescence
(excitation: 587 nm; emission maximum: 610 nm).
5. Day 4: Collect the culture supernatant 96 h after infection by
centrifugation for 15 min at 6000 Â g at 4
C. Discard the cell
pellet. Adjust the pH of the supernatant to ~8.0 by adding
1/20 volume of 1 M Tris-HCl (pH 8.0).
3.2.3 Purification
of Genetically Encoded
Fluorescent Fab Fragments
1. Day 1: (IMAC) Use 20 mL of Ni Sepharose excel resin (40 mL
of 50% slurry) per 1 L of Sf9 culture, pour the slurry into a glass
Econo-Column, and equilibrate with five CVs of buffer A.
2. Add the Sf9 culture supernatant directly to the column, and
collect the flow-through fraction.
3. Wash the column with 10 CVs of buffer A.
4. Elute the polyprotein of interest from the column by adding
three CVs of buffer B and collect the eluate.
5. Measure the protein concentration of the eluate using a NanoDrop spectrophotometer.
6. Concentrate the protein using an Amicon Ultra-15 (MWCO
30 K) centrifugal concentrator to the volume of 5 mL.
7. (SEC) Inject the 5 mL of concentrated protein sample onto a
HiLoad16/60 Superdex200 column pre-equilibrated with
TBS at a flow rate of 1 mL/min.
Production of Recombinant Antibody Fragments via the iRAT system
97