Expression levels and labeling efficiency of ptfV Cys variants
| Mutation position . | fV domain . | BHK-M stable expression, mg/L media . | PEGylation efficiency, %* . |
|---|---|---|---|
| K134C | A1 | 1.95 | 49 |
| E168C | A1 | 1.65 | 72 |
| T276C | A1 | 2.27 | 76 |
| C221A (C302) | A1 | 0.38 | 39 |
| R310C | a1 | 1.26 | 65 |
| K311C | a1 | 0.90 | 71 |
| L312C | a1 | 2.89 | 75 |
| F314C | a1 | 2.01 | 85 |
| R315C | a1 | 4.38 | 85 |
| E316C | a1 | 4.83 | 50 |
| K321C† | a1 | NA | NA |
| T378C† | A2 | NA | NA |
| T403C | A2 | 4.63 | <6 |
| V420C | A2 | 3.55 | <15 |
| N441C | A2 | 1.66 | 83 |
| T455C | A2 | 2.93 | 27 |
| C540‡ | A2 | 3.98 | 59 |
| C540A§ | A2 | 2.09 | ND |
| S578C | a2 | 2.76 | 36 |
| Mutation position . | fV domain . | BHK-M stable expression, mg/L media . | PEGylation efficiency, %* . |
|---|---|---|---|
| K134C | A1 | 1.95 | 49 |
| E168C | A1 | 1.65 | 72 |
| T276C | A1 | 2.27 | 76 |
| C221A (C302) | A1 | 0.38 | 39 |
| R310C | a1 | 1.26 | 65 |
| K311C | a1 | 0.90 | 71 |
| L312C | a1 | 2.89 | 75 |
| F314C | a1 | 2.01 | 85 |
| R315C | a1 | 4.38 | 85 |
| E316C | a1 | 4.83 | 50 |
| K321C† | a1 | NA | NA |
| T378C† | A2 | NA | NA |
| T403C | A2 | 4.63 | <6 |
| V420C | A2 | 3.55 | <15 |
| N441C | A2 | 1.66 | 83 |
| T455C | A2 | 2.93 | 27 |
| C540‡ | A2 | 3.98 | 59 |
| C540A§ | A2 | 2.09 | ND |
| S578C | a2 | 2.76 | 36 |
NA, not applicable; ND, none detected.
Small-scale labeling was conducted with maleimide-PEG40K. All variants were run on 3% to 8% Tris-acetate gel postlabeling with maleimide-PEG40K (supplemental Figure 1). Percentage of PEGylation was estimated by comparing the PEGylated band with unPEGylated band on SDS-PAGE by densitometry.
BHK-M cells were transfected 3 times and stable cells selected; however, no expression was observed.
P textilis fV has 15 cysteine residues in total, 14 of which form disulfide bonds. C540 is a solvent-exposed free cysteine at the back of the A2 domain.
C540A mutation was generated to see if labeling is specific for cysteine residues. All mutations to introduce single cysteine residues were made on this background.