Elementary Study On The Protein Cross-Linking Of Recombinant Escherichia Coli NADP-specific Glutamate Dehydrogenase In Vitro

Protein interchain cross-linking is a common phenomenon in the living creatures and are closely related with many physiology and pathologic processes. However the molecular mechanisms of proteins from monomeric/functional forms into cross-linked dimmer/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 cross-linking can happen in heterogeneous peptides, the investigation is extended with E.coli NADP-specific glutamate dehydrogenase (NADP-GDH). NADP-GDH gene was cloned by PCR from E.coli DH_5a, and inserted into plasmid pTrcHisC for over expression in E.coli. Then it was purified by Immobilized Metal Affinity Chromatography with the help of (His)6 tag. At last NADP-GDH and lysozyme was 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 dimmer/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 conformed the three-step hypothesis of protein cross-linking.