Strain | Genotype | Source | Nanobody A | Nanobody B | Nanobody C |
---|
Titers (g l−1) | % Δ−16 Da variant | Titers (g l−1) | % Δ−16 Da variant | Titers (g l−1) | % Δ−16 Da variant |
---|
CBS7435 (NRRL Y-11430) |
AOX1
| ARSa
| 5.2 | 0 | 7 | 0 | | |
CBS7435MutS
|
aox1
| [7] | 4.5 | 4 | 10.5 (2.5)a
| 12 (0)a
| | |
X-33 |
AOX1
| Invitrogen | 6.4 | 0 | 9.1 | 0 | 0.9 | 0 |
KM71H (MutS) |
aox1
| Invitrogen | | | | | 1.2 | 8 |
- Nanobody productions were performed at 2 l scale, pH 6, 30 °C in complex medium with a methanol feed rate of 4 or 3 ml l−1 h−1 for wild type or MutS strains respectively. Except for a where the methanol feed rate was reduced to 0.5 ml l−1 h−1 in a co-feeding with sorbitol. Expression levels of Nanobodies were analyzed via a proteinA cleanup step followed by OD280 measurement. Relative abundance of the Δ−16 Da variant of the different Nanobodies was analysed via RP-HPLC followed by total mass measurement by ESI-Q-TOF-MS. The Δ−16 Da Nanobody variant was only observed in fed-batch fermentations with the MutS strains. A strong reduction of the Δ−16 Da was observed when the methanol feed rate was reduced to 0.5 ml l−1 h−1 and using co-feeding with sorbitol