Table 3.
Attempts to introduce a stabilizing disulfide bond at different locations in RTA1-33/44-198.
| Ricin vaccine immunogen | Immunogen | Sequencing 1 | Solubility/Purity 2 | Number of free Cys 3 | Apparent Tm | Disruption of VLP? |
|---|---|---|---|---|---|---|
| Purification Method No. 1 | ||||||
| RTA | - | Yes | S+/>95% | - | 53.5 ± 0.74 | No |
| RTA1-33/44-198 | - | Yes | S+/>95% | - | 61.0 ± 0.23 | No |
| RTA1-33/44-198 | D75N | Yes | S+/>95% | - | 60.0 ± 0.05 | Yes |
| RTA1-33/44-198 | V76I | Yes | S+/>95% | - | 59.8 ± 0.17 | Yes |
| RTA1-33/44-198 | D75A | Yes | S+/>95% | - | 56.6 ± 0.10 | Yes |
| RTA1-33/44-198 | V76M | Yes | S+/>95% | - | 55.4 ± 0.42 | Yes |
| Purification Method No. 2 | ||||||
| RTA1-33/44-198 | - | Yes | S+/>95% | 0.97 ± 0.03 | 57.9 ± 0.03 | No |
| Substitutions that Increase Apparent Tm | ||||||
| RTA1-33/44-198 | R48C/T77C | Yes | S+/>95% | 1.00 ± 0.08 | 62.9 ± 0.22 | No |
| RTA1-33/44-198 | V49C/E99C | Yes | S+/>95% | 0.96 ± 0.05 | 62.9 ± 0.21 | No |
| RTA1-33/44-198 | R48C/T77C/D75N | Yes | S+/>95% | 1.04 ± 0.02 | 63.2 ± 0.26 | Yes |
| RTA1-33/44-198 | V49C/E99C/V76I | Yes | S+/>95% | 0.99 ± 0.03 | 62.6 ± 0.21 | Yes |
| RTA1-33/44-198 | V49C/E99C/D75N | Yes | S+/>95% | 1.10 ± 0.06 | 62.2 ± 0.13 | Yes |
| RTA1-33/44-198 | R48C/T77C/V76I | Yes | S+/>95% | 1.89 ± 0.03 | 59.3 ± 0.60 | Yes |
1 Presence of desired substitution and no other was confirmed by DNA sequencing; 2 Solubility was assessed qualitatively by evaluating Coomassie blue stained SDS-PAGE gels of initial induction checks. Cells over-expressing recombinant protein were fractionated and scored as I (>50% detected in the insoluble pellet), S (>50% in soluble fraction), or S+ (entirely in soluble fraction). Purity of final product was estimated from Coomassie blue-stained SDS-PAGE. The identity of purified protein was confirmed by reaction with antisera on Western blots; 3 The number of free Cys residues was quantified for purified proteins by titrating free –SH using 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB). DTNB titrations were conducted in triplicate at four different protein concentrations between 2 and 11 μM; the mean ± SE was determined from linear regression of the titration plots. Note that the parent protein molecule (RTA1-33/44-198) contains one Cys, whereas each mutant contains a total of three Cys residues. The DTNB assay was optimized to probe the reduced Cys residues such that values significantly greater than one reflect incomplete formation of a stable disulfide bond between the engineered Cys residues.