Figure 4.

Contributions of a critical residue to the mAb 2E4 epitope. (a-1) 3D model of the F1 fragment (in red). (a-2) Display of 2α-helical structures on the F1 fragment (in purple and yellow). (a-3) All amino acid residues of interest in the 2α-helical structures. (b) Approximately equal amounts of E. coli cell lysates with the expressed GST-fusion proteins were electrophoresed on a 12% SDS-PAGE gel and stained with Coomassie blue. 6p-1 represents the pGEX-6p-1 vector; K⁶¹, K⁶⁹, K⁷⁵, D⁹⁴, E⁹⁵, and L⁹⁶ represent individual mutants in which the indicated amino acids were substituted with alanines; “All” represents the mutant in which K⁶¹, E⁶², D⁹² and D⁹⁴ were simultaneously substituted. (c) Expression products are exhibited at approximately 28 kD. The purified expression products were in accord with the situation in (b) and used to investigate the reactivity of the variants if the interesting residues were deficient. (d) Individual substitutive mutants with reactivity to mAb 2E4 were evaluated by ELISA. The pGEX-6p-1 vector was used as a negative control. Only D⁹⁴ showed remarkably attenuated reactivity to 2E4, contrasting with F1 and other mutant objects. (e) “ALL” fully abolished the reactivity of the epitope to 2E4, in contrast with the D⁹⁴ mutant. F1 is a positive control in this test (*p < 0.05, represents a statistically significant difference; **p < 0.01, represents a statistically highly significant difference; Mean ± SD, n = 3).