Product analysis
For SpA quantification, 10 mL culture were centrifuged for 10 minutes at
15,000 rcf and 4°C. The supernatant was aliquoted and stored at -20°C.
The cell pellet was re-suspended in 35 mL of TRIS-buffer (100 mM TRIS,
10 mM EDTA, pH 7.4). This suspension was homogenized in an Emulsiflex C3
homogenizer (Avestin, Ottawa, ON, Canada) (5 passages, 1000 bar) and the
sample was then centrifuged for 15 minutes (20,000 rcf, 4°C). The pellet
was stored at -20°C. Intracellular soluble SpA content and SpA content
in the cell-free culture supernatant were quantified in triplicate by
HPLC analysis using a reversed phase column (BioResolve™ RP mAb
Polypheyl; Waters, Milford, MA) and a gradient of acetonitrile and
water, both supplemented with 0.1% (v/v) trifluoroacetic acid.
VHH quantification was done analogously, with the exception that the
cell pellet was sonicated in MES-Buffer (100 mM MES, 10 mM EDTA, pH 6.0)
and HPLC analysis was performed with a cation exchange column
(BioResolve™ SCX mAb; Waters). The loading buffer was 20 mM MES, pH 6.0
and VHH was eluted with a Na+ gradient.
Inclusion body formation of VHH was analyzed qualitatively by SDS-PAGE.
For this, the pellet obtained after homogenization was resuspended in 20
mL of Buffer A (50 mM TRIS, 0.5 M NaCl, 0.02% Tween, pH 8.0) and then
centrifuged for 10 minutes (10,000 rcf, 4°C). The resulting pellet was
washed in 20 mL Buffer B (50 mM TRIS, 5 mM EDTA, pH 8.0) and 2 mL
aliquots were centrifuged for 10 minutes (10,000 rcf, 4°C).
Subsequently, the pellet was resuspended in 1 mL ultrapure water,
diluted with 1.5 × Laemmli buffer. A VHH standard (5 g/L) was diluted in
2 × Laemmli buffer. The samples and standard were then incubated at 95°C
for 15 minutes. 10 µL of sample and 5 µL of standard were loaded onto
precast SDS gels (8-16%, Mini-PROTEAN TGX; Bio-Rad, Hercules, CA). Gels
were run at 120 V for 30 minutes in a Mini-PROTEAN Tetra-Cell (Bio-Rad)
and stained with Coomassie Blue. Images were captured and analyzed using
the software Image Lab (Bio-Rad).