title: Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV.
abstract: 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 in-vivo 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.
authors.affiliation: GBF National Research Center for Biotechnology, Biochemical Engineering Division, Braunschweig, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Oswald
authors.forename: W
authors.initials: W
authors.lastname: Wende
authors.forename: A
authors.initials: A
authors.lastname: Pingoud
authors.forename: U
authors.initials: U
authors.lastname: Rinas
mesh_terms: D001709:Biotechnology; D002846:Chromatography, Affinity; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases