Manipulation of Determinants
The design and alteration of epitope tags and the genes they highlight has been streamlined and expedited through an array of commercial technologies. According to a number of scientists interviewed for this article, the Gateway™ technology (invitrogen.com/gateway) from Invitrogen (www.invitrogen.com) has facilitated the manipulation of epitope tags. Gateway is a universal cloning technology that allows the transfer of DNA segments through a variety of cloning vectors with simplicity and high efficiency. This feat is accomplished through use of the attP integration site in Phage Lambda and its partner bacterial integration site attB in E. coli.
The gene of interest is first cloned into an archival vector (Entry vector), which contains attL recombination sites just upstream and downstream of the inserted gene. From here, the gene fragment, or ORF, can be moved into any vector containing attR site (Destination vector). Destination vectors typically contain upstream promoters required to initiate transcription in specific hosts and epitope tags either at the 5´ or 3´ ends of the gene.
The protocol is fast and simple; to subclone the gene of interest from the entry clone to the destination vectors, the two are mixed together and incubated in the presence of the cloning enzyme mixture for one hour. The company supplies a range of destination vectors with various epitope tags for use in in vitro, cell-free E. coli, yeast, insect, mammalian, adeno, and lentiviral systems.
Hundreds of other Destination vectors have been developed by academic laboratories across the world. “Currently, Invitrogen is the only commercial provider of Gateway Destination vectors,” reports Balwant Patel, Ph.D., business area manager for cloning and expression products and services at Invitrogen.
Invitrogen recently extended a license to Covalys Biosciences (www.covalys.com) for the commercialization of Destination vectors containing the SNAP-tag technology for specific protein labeling in living cells. According to Dr. Patel, Invitrogen is in the process of liberalizing its licensing policy and is open to discussions with other life-science providers who may be interested in supplying their own Gateway Destination vectors to researchers in academia and industry.
The technology takes advantage of two types of expression vectors, supporting either the expression of untagged proteins or fusion proteins. For the various vectors, different expression elements may need to be installed between the attB sites prior to the Gateway reactions.
The basic Gateway technology, available since 1999, has been improved to allow for simultaneous cloning of multiple DNA fragments. The Multisite Gateway Kit and Pro technologies enable the assembly of as many as four elements in a single plasmid in correct order and orientation. This is accomplished by increasing the number of available att recombinational sites. These modifications of the original Gateway technology make it quite useful for the expression of proteins with different fusion partners and epitope tags.