A bifunctional fusion protein containing the maltose-binding polypeptide and the catalytic chain of aspartate transcarbamoylase: assembly, oligomers, and domains

Abstract

The in vivo synthesis of many target proteins or polypeptides has been enhanced dramatically and their purification facilitated through the use of gene fusion techniques which lead to the expression of fusion proteins. This approach was used to characterize the product formed in Escherichia coli encoded by a DNA construct comprising malE, the gene encoding maltose binding protein, linked to a small 30 nucleotide region which, in turn, was linked to pyrB, the gene encoding the catalytic (c) chains of aspartate transcarbamoylase (ATCase). The resulting fusion protein, MBP-C, was produced in excellent yield and readily purified in two steps because of its binding to an amylose column and displacement by maltose. The complex was studied by both sedimentation velocity and sedimentation equilibrium and shown to be a trimer of c chains with one MBP linked covalently to each chain. Treatment of the fusion protein with factor Xa cleaved each chain at the tetrapeptide encoded by the linker region yielding purified MBP with a minor modification at the C-terminus and the catalytic (C) trimer of ATCase. The MBP-C complex was fully active as an enzyme and could be reversibly denatured in 6 M urea. Scanning calorimetry studies on the fusion protein demonstrated that the MBP domain melted at the same temperature as did the purified protein. Similarly, the T m for the C trimer in the complex was identical to the value for C trimer isolated from ATCase. Moreover, the thermal stability of the C trimer in the MBP-C complex was greatly enhanced by the addition of the bisubstrate ligand, N-(phosphonacetyl)- l-aspartate (PALA), just as observed with purified C trimer. Analogous denaturation experiments with varying concentrations of guanidine-HCl indicated that the fusion protein was denatured at much lower concentration of denaturant than observed for C trimer. These experiments demonstrate that the linker between the two structural genes encodes a polypeptide of sufficient length to permit independent folding and assembly of each protein and permit the subsequent specific cleavage at the factor Xa recognition site, thereby yielding both active proteins.