Protein Cross-Linking During Thermal Unfolding Of Lysozyme And Recombinant Escherichia Coli Protein

The Escherichia coli thioredoxin (TRX) gene and ribonucleic acid enzyme Hâ…¡ (RNase Hâ…¡) gene, cloned by PCR, were inserted into plasmid pTrcHisC for over expression induced by IPTG; the recombinant proteins were purified by using TALON metal affinity resin. Thermal unfolding of recombinant Escherichia coli thioredoxin (TRX), ribonucleic acid enzyme Hâ…¡ (RNase Hâ…¡ ), and Lysozyme led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis; in addition, heterogeneous proteins also cross-linked. Preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide bond cross-links, because no dimers/oligomers were detected when the unfolding solution contained the reducing agent dithiothreitol. Furthermore, the Cys334Ser point mutation in ribonucleic acid enzyme Hâ…¡ abrogated its ability to cross-link into homodimers. All these suggested that protein cross-linking can be accomplished in three concerted steps: (i) changes in protein conformation; (ii) formation of inter-molecule disulfide bonds; and (â…¢) formation of inter-molecule isopetide cross-linking.