Figure 4

Purification of hRIα subunit with different Sp-cAMPS analog agaroses analysed by SDS-PAGE. (A) crude: total cell extract of E. coli BL21 DE3 Codonplus RIL with overexpressed hRIα at 52 kDa; supernatant: soluble fraction of bacterial lysate after French Press and centrifugation; pellet: insoluble fraction; sup agarose: unbound protein after incubation with Sp-8-AEA-cAMPS agarose; wash agarose: aliquot after 6 washing steps with buffer B; elution cGMP and elution cAMP: elution with 10 mM cGMP and subsequently with 10 mM cAMP, respectively; wash agarose: aliquot after 6 washing steps with buffer B; agarose beads after elution: remaining protein on the agarose beads after the two elution and washing steps. M: molecular weight marker (Page-Ruler Unstained Protein Ladder, Fermentas). (B) Total cell extract of E. coli BL21 DE3 Codonplus RIL with overexpressed hRIα was incubated with Sp-2-AHA-cAMPS and Sp-8-AHA-cAMPS, respectively, and eluted with cGMP (10 mM, lane 1 and 4) and subsequently with cAMP (10 mM, lane 2 and 5). Only a low amount of protein was left on Sp-2-AHA-cAMPS agarose after elution with cAMP (lane 3), whereas significant amounts of R-subunit remained on Sp-8-AHA-cAMPS agarose (lane 6). Asterisk indicates Chloramphenicol Acetyltransferase from E. coli BL21 (DE3) Codon Plus RIL as identified by MS.