title: Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins.
abstract: Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three mini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novel intein fusion system. In this system, green fluorescent protein (GFP) was fused 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 monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins 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 soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications.
authors.affiliation: New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
authors.forename: A
authors.initials: A
authors.lastname: Zhang
authors.forename: S M
authors.initials: SM
authors.lastname: Gonzalez
authors.forename: E J
authors.initials: EJ
authors.lastname: Cantor
authors.forename: S
authors.initials: S
authors.lastname: Chong
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004258:DNA Polymerase III / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics; D011499:Protein Processing, Post-Translational; D019154:Protein Splicing; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D009841:Oligonucleotides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; D004247:DNA; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III