Cloning, Expression, Purification And Protein Cross-linking Of Escherichia Coli Dihydrofolate Reductase

Interchain isopeptide cross-link is a common kind of molecular cross-links in proteins. Researches about it are focus on transglutaminases and Ubiquitin-proteasome pathway. By using lysozyme, RNase A and protein disulfide isomerase (PDI) as model proteins, we present a hypothesis that 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.By using the Polymerase Chain Reaction (PCR) technique, the 0.49kb DNA fragment of dihydrofolate reductase (DHFR) gene (folA) was amplified from the Escherichia coli DH5. cell lysate. The fragment was inserted into plasmid pUC18 by the sites of two restriction enzyme BamH I and Pst I, and the target gene was confirmed by DNA sequencing. Then the target gene was subcloned into the expression plasmid pTrcHisC. The recombinant protein expression was induced by IPTG The recombinant DHFR protein containing his-tag was purified by using Talon Metal Affinity Resin under nondenaturing conditions. Under the thermal unfolding conditions, dimer/polymer bands in addition to the major 23 kDa band were observed on the SDS-polyacrylamide gel. In contrast, the dimer/polymer was disappeared when reaction solution contained reducer dithiothreitol(DTT). We concluded that protein cross-linking involved in a change in protein conformation and the formation of interchain disulfide bonds.By using the Polymerase Chain Reaction (PCR) site mutation technique, The mutant (Cys85/152Ser) DNA fragment of Escherichia coli dihydrofolate reductase gene was amplified. The fragments were inserted into the expression plasmid pTrcHisC, The recombinant protein expression was induced by IPTG and purified. In contrast, the Cys85/152Ser mutant completely failed to form dimers under the same conditions. The results again substantiate that to form an interchain disulfide bond is critical in promoting subsequent protein cross-links.DHFR and lysozyme were mixed during thermal unfolding. The observation revealed that heterodimer/oligomer formation was as common as homodimer/oligomer formation.