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. 2020 Oct 26;48(22):e128. doi: 10.1093/nar/gkaa933

Figure 2.

Figure 2.

Coupling of Sso7d and Stoffel fragment mediated by SpyCatcher-SpyTag interaction facilitates PCR in high-salt buffer conditions. (A) Indicated proteins were (co)-expressed in Escherichia coli and cells directly used in PCR reactions with increasing KCl concentrations (0, 100, 200, 300 mM). S7d-S: Sso7d-Stoffel fusion; S7d-SC: Sso7d-SpyCatcher fusion; ST-S: SpyTag-Stoffel fusion; S: Stoffel; n=1. Replicate data shown in Figure 1C for S7d-S lanes and 3B, 7B for Sd7-SC + ST-S and S7d-SC + S lanes. (B) SDS-PAGE analysis of uninduced/induced E. coli cell lysates (co)-expressing indicated proteins. Highlighted bands indicate 1: Sso7d-Stoffel fusion (S7d-S); 2: Stoffel fragment (S); 3: Sso7d-SpyCatcher fusion (S7d-SC); 4: Sso7d-SpyCatcher fusion conjugated to SpyTag-Stoffel fusion (ST-S); 5: SpyTag-Stoffel fusion (ST-S); 6: Sso7d (S7d); n=1. Replicate data for S7d-SC + S and S7d-SC + ST-S lanes shown in Figure 7A. (C) Indicated proteins (recombinantly expressed and purified) were (co)-incubated for 30 min and an aliquot used in PCR with both normal and high-salt buffer (+ 100 mM KCl). The same reaction mixes were analyzed by SDS-PAGE (right). Highlighted band (*) indicates S7D-SC-ST-S fusion protein; n=1.