Table 2. CB-EGF.2 mAb-driven selection of the original residues from a library of EGF mutated variants.
| Targeted segment | Original residue | Unselected phage-displayed mAb- negative EGF variants | Selected mAb-positive EGF variants |
|---|---|---|---|
| M21-L26 | M21 |
5.0% Met other: ACEGHIKPQRST |
No Met other: ADEGHIKLNQRSTVWY |
| Y22 |
5.0% Tyr other: ADEFKLMPRSTV |
44.1% Tyr (> 8x) other: AFHILMNQRST |
|
| I23 |
No Ile other: DGHLPQRSTY |
25.6% Ile (> 25x) 69.8% hydrophobic: ILV other: AHKQRSW |
|
| E24 |
No Glu other: ACDFGHILMNPTV |
7.0% Glu (> 7x) other: AHIKLMNPQRSTVY |
|
| A25 |
15% Ala other: EFGHILPQST |
27.9% Ala other: HLMPQVY |
|
| L26 |
No Leu other: ADGHKMPQRVWY |
69.8% Leu (> 69x) 100% hydrophobic: ILMV no other residues |
|
| D27-N32 | D27 |
4.2% Asp other: AEFILPQRST |
60.5% Asp (> 14x) other: AGNS |
| K28 |
No Lys other: AGHNPQRST |
32.6% Lys (> 32x) 76.8% positively charged: KR other: LMNSTV |
|
| Y29 |
No Tyr other: ACEGHIKPQST |
4.7% Tyr (> 4x) other: ADEGHIKLMNPQRST |
|
| A30 |
No Ala other: EHMNPQRSTV |
14.0% Ala (> 14x) other: DEFGHKLMNQSTVWY |
|
| N32 |
4.2% Asn other: ACDEGHPQRSTV |
4.7% Asn other: ADELMPQRSTY |
Residues within the M21-L26 and D27-N32 segments were replaced by random aa mixtures in two separate libraries. Samples of non-selected CB-EGF.2-negative EGF variants and positive variants selected on CB-EGF.2 mAb were sequenced. The frequencies (%) of each original residue in both groups of variants are shown. The relative increase in the frequency of a given residue after selection is indicated between parentheses. The combined prevalence (%) of residues with shared physicochemical properties at some positions is represented. Other amino acids found at each position in both groups of variants are also shown. Residues that contribute to epitope formation are shaded in gray, whereas those classified as major functional contributors are highlighted in dark gray.