Elementary Study On The Purification And Protein Cross-linking Of Human Peptidylprolyl-cis-trans-isomerase Cyclophilin

Protein interchain cross-linking is a common phenomenon. It has been implicated in several physiological and pathological processes. However the molecular mechanisms for converting monomeric/functional forms of proteins into cross-linked dimer/oligomer forms are not well understood. Experiments with lysozyme, ribonuclease A and protein disulfide isomerase in vitro show protein cross-linking can be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links. For further exploring if the three-step hypothesis of protein cross-linking is right, and if cross-linking can happen hi heterogeneous peptides, the investigation is extended with human peptidylprolyl-cis-trans-isomerase cyclophilin (hPPI cyclophilin). hPPI cyclophilin cDNA was cloned by RT-PCR from Chinese human fetal liver, and inserted into plasmid pTrcHisC for overexpression in E. coli. Then it was purified by Immobilized Metal Affinity Chromatography with the help of 6*His affinity tag. At last, hPPI cyclophilin and lysozyme were mixed during thermal unfolding. The observation revealed that heterodimer/oligomer formation was as common as homodimer/oligomer formation in the midpoint temperature of thermal denaturation. However no cross-linked dimer/oligomer appeared in low temperature. If the thermal unfolding solution contained the reducing agent P -Mercaptoethanol, no cross-linking was detected. It was apparent that cross-linking can't happen until the protein secondary structure had changed to a certain extent, and that preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links. All these results confirmed the three-step hypothesis of protein cross-linking.