Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins

Affinity purification of recombinant proteins has been facilitated by fusio n to a modified protein splicing element (intein). The fusion protein expre ssion can be further improved by fusion to a mini-intein, i.e. an intein th at lacks an endonuclease domain. We synthesized three rnini-inteins using o verlapping oligonucleotides to incorporate Escherichia coli optimized codon s and allow convenient insertion of an affinity tag between the intein (pre dicted) N- and C-terminal fragments. After examining the splicing and cleav age activities of the synthesized mini-inteins, we chose the mini-intein mo st efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was f used to the C-terminus of the affinity-tagged mini-intein whose. N-terminus was fused to a target protein. The design of the system allowed easy monit oring of soluble fusion protein expression by following GFP fluorescence, a nd rapid purification of the target protein through the intein-mediated cle avage reaction. A total of 17 target proteins were tested in this intein-GF P fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion pro teins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence, The intein-GFP system may provide a simple route for monitoring real time. sol uble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purificatio n in high throughput applications. (C) 2001 Elsevier Science B.V. All right s reserved.