COMPARISON OF N-TERMINAL AFFINITY FUSION DOMAINS - EFFECT ON EXPRESSION LEVEL AND PRODUCT HETEROGENEITY OF RECOMBINANT RESTRICTION-ENDONUCLEASE ECORV

The influence of different N-terminal affinity fusion domains on the p roduct heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction en donuclease EcoRV with either glutathione-S-transferase [GST], histidin e hexapeptide [(His)(6)], or a combination of GST and (His)(6) [GST-(H is)(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-dimens ional (2-D) non-equilibrium pH-gradient electrophoresis with subsequen t immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highl y susceptible to invivo aggregation and fragmentation and displayed mo re heterogeneity on 2-D immunoblots. However, the sole presence of oli gohistidine at the N-terminus of EcoRV proved to be advantageous. Frag mentation of (His)(6)-EcoRV was not observed and 2-D immunoblots did n ot show heterogeneous forms of the recombinant protein. In addition, f usion of the histidine-hexapeptide to the N-terminus of native EcoRV i ncreased the expression level of the recombinant protein twofold compa red to native EcoRV. Inclusion body formation of the (His)(6)-EcoRV fu sion protein was intensive when cells were grown at 37 degrees C but n ot at 30 degrees C. The advantage of oligohistidine fusion to EcoRV wa s finally demonstrated by purifying soluble (His)(6)-EcoRV in a single -step procedure from crude cell lysates using immobilized metal chelat e affinity chromatography.