2.4. Preparation of ORF8-CRM and ORF8-PEG
ORF8 (2 mg) was incubated with 80-fold molar excess of IT to introduce the thiol groups in PBS buffer (pH 7.4) at 4°C for 8 h (Fig. 1). CRM197 (2 mg) was incubated with 80-fold molar excess of EMCS to introduce the maleimide groups in PBS buffer (pH 7.4) at 4°C for 6 h (Fig. 1). The free IT and EMCS were removed by extensive dialysis against PBS buffer (pH 7.4). Then, the ORF8-CRM197conjugate (ORF8-CRM) was obtained by incubating the thiol groups of ORF8 and the maleimide groups CRM197 at 4°C for overnight.
ORF8 (2 mg) was reacted with 10-fold molar excess of 8-arm PEG in PBS buffer (pH 7.4) at 4°C for overnight to obtain the conjugate (ORF8-PEG).