Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags.
We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.
Gillies
Alison R
AR
Department of Chemical Engineering, Princeton University, A417 Engineering Quadrangle, Olden Street, Princeton, New Jersey 08544, USA.
Hsii
Judy F
JF
Oak
Seachol
S
Wood
David W
DW
D002374:Catalase / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D005822:Genetic Vectors; D047668:Inteins; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification*; D001618:beta-Lactamases / Q000302:isolation & purification
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D002374:Catalase; C051253:katG protein, E coli; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1