We have characterised the stability, binding and enzymatic properties of three human FK506 binding proteins (FKBP-12) differing only by the length and sequence of their N-terminus. One construct has a short hexa-his tag (6H-FKBP12); the second longer fusion protein (6HL-FKBP12) contains an additional thrombin protease cleavage sited the third has the long fusion tag removed and is essentially native FKBP-12 (cFKBP12). The proteins were purified both under native conditions and also using a refolding protocol. All three natively purified proteins have, within experimental error, the same peptidyl-prolyl isomerase (PPIase) activity (k(cat)/K-m approximate to 1 X 10(6) M-1 s(-1)), and bind a natural inhibitor, rapamycin, with the same high affinity (K-d approximate to 6 nM). However, refolding of the protein containing the longer tag in vitro results in reduced PPIase activity (the k(cat)/K-m was reduced from I X 10(6) M-1 s(-1) to 0.81 X 10(6) M(-1)s(-1)) and a 6-fold affinity loss for rapamycin. Addition of both the long and short N-terminal his-tags slows the refolding kinetics of FKBP-12. However, the shorter his-tagged fusion protein regains fully native activity ( >= 95%) while the longer regains only similar to 80-85%, of native activity. Equilibrium urea denaturation titrations, isothermal titration calorimetry {ITC), analytical gel-filtration, and fluorescence binding data show that this loss of activity is not due to gross misfolding events, but is rather caused by the formation of a stable but subtly misfolded protein that has reduced peptidyl-prolyl isomerase (PPIase) activity and reduced affinity for rapamycin. The difference in behaviour between the in vitro refolded and native forms is due to the dominant role of the cellular chaperone/folding machinery. (c) 2006 Elsevier Inc. All rights reserved.