Fig. 5

Production and Purification of native RTAM-PAP1. a Loosely bound proteins were washed with the lysis buffer containing 50 mM imidazole (I50) on a Ni-sepharose column and RTAM-PAP1 (RPAP1) proteins were then eluted with the elution buffer containing 300 mM Imidazole (I300). b The Western Blot using ricin a chain antibody RA999 confirmed the presence of RTAM-PAPS1 at approx. 61.5 kDa. The bands between 21 kDa and 32 kDa are assumed to be degraded or/and premature RTAM-PAP1 proteins. c (Lys) from 1 L culture. a) Loosely bound proteins were washed with the lysis buffer containing 40 mM imidazole (I40) on a Ni-sepharose column and RTAM-PAP1 proteins were then eluted with the elution buffer containing 300 mM Imidazole (I300). d Co-purified host cell proteins were further separated by a hydroxylapatite column. Most RTAM-PAP1 proteins were retained in the flow through (FT) fraction, while most host cell proteins were bound to the hydroxylapatite column (P200 elution). e RTAM-PAP1 was peaked at fraction 15 and 16. The purest fraction (F15) was estimated at > 95% homogeneity (f) and was used for the inhibition assay