The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)(6)], or a combination of GST and (His)(6) [GST-(His)(6)] were compared to native EcoRV with respect to expression level, susceptibility to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non-equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)(6)-EcoRV was not observed and 2-D immunoblots did not show heterogeneous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)(6)-EcoRV fusion protein was intensive when cells were grown at 37 degrees C but not at 30 degrees C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)(6)-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.