Fig. 2

Sequence modification to enhance physicochemical properties. a EBOV-GP-A8-neg+ sequence has four amino acid substitutions within FR1 (Q5V, A6E, A13V, and G17D), while EBOV-GP-G6-neg+ in b and EBOV-GP-H7-neg+ in c have only two substitutions in FR1, Q5V, A6E. All of the mutants have an insertion of a disulfide bond formed by two substituted Cys, A54C and I78C, within FR2 and FR3 indicated in a-c. EBOV-GP-A8-fneg+ sequence in a has the same sequence as EBOV-GP-A8-neg+ except with replaced C106S, which eliminates an unpaired Cys thus disrupting the potential formation of an inter-disulfide bond. d The underlined GGGGSGGGGKKK (GSKKK) sequence in bold font is fused onto the C-terminus of EBOV-GP-A8-fneg+, EBOV-GP-G6-neg+, and EBOV-GP-H7-neg+. Following each clone name the suffix “neg” indicates mutations in FR1 that result in the addition of negative charges, and the “+” indicates the addition of one disulfide bond formed by two substituted Cys in FR2 and FR3. The “f” denotes “fixing” the EBOV-GP-A8 sdAb through mutating an unpaired Cys to Ser in FR3 as indicated in a