Multi-Gene/Protein Expression Systems

Multi-protein assemblies dominate a slew of biological processes such as transcription, translation, RNA splicing, signaling cascades, protein transport, trafficking and degradation. Life science researchers have become increasingly interested in studying these complexes in more detail as they realize that no protein works in isolation and thus its function needs to be studied in its cellular context.

Another avenue where such protein clusters become important is in industrial biotechnology/metabolic engineering, i.e. when trying to coax microbes into producing complex, valuable substances, to tune them for bio-remediation, and other processes that require changing the biochemical behavior of these cells.

For a long time, co-transfection was the method of choice to insert foreign genes into the chosen organism. Gradually, more sophisticated technologies for the transfer of multiple genes/proteins have been developed, along with ways of maintaining them stably over long periods in the host.

Multi-gene constructs for multi-protein and multi-peptide expression:
Protein and peptide expression are centerpieces of experimental molecular biology that allow generation of bulk amounts of proteins that may otherwise occur only spuriously in the cell. These proteins may be generated in their native hosts but more commonly are over-expressed in heterologous hosts that are easier to genetically modify than the donor organism.

ATG: biosynthetics can provide all the key ingredients needed for multi-gene expression:

• a plasmid-based system (or alternative) to build your constructs
• biocomputing expertise to allow adaptation of gene expression parameters to the target host
• Relevant design pointers

ATG offers a series of systems to handle these multiple protein and gene expressions and constructions. The first one is MultiLabel which can be used to transiently or stably introduce multiple proteins into mammalian cells.

MultiLabel
ATG: biosynthetics offers a multi-protein expression technology, MultiLabel, that works for up to five proteins expressed simultaneously. It therefore offers a solution to what has long been a challenging problem – co-expression of multiple proteins in mammalian cells.

It provides a greatly superior mechanism than traditional co-transfection, in which the likelihood of getting all desired plasmid construct into any given cell decreases with the number of plasmids being transferred.

MultiLabel is a suite of vectors designed specifically for multi-protein expression in mammalian cells. This allows genes of interest ito be cloned in any of the acceptors and donors and generated as multi-gene assemblies by recombination (see workflow diagram).

Three acceptor and two donor vectors are currently available, all equipped with the strong eukaryotic CMV promoter and a bovine growth hormone polyadenylation signal. Acceptors come with a standard ori for replication in standard laboratory strains whereas donors sport the R6K gamma ori that allows replication only in strains expressing the pi protein (pir-positive strains). This concept has been proven in other systems ATG has distributed for many years.

Applications:
MultiLabel can be used :
• to generate multigene expression plasmid constructs for transient or stable expression
• for structural investigations, protein tracking, elucidation of signal cascades, etc.
• for cell line development (sensors, etc.) or cellular reprogramming
• for generating transgenic animals or cell lines
• for cell-free translation

For more information on our multi-gene/protein expression systems or to discuss how they can benefit your processes, please contact ATG:biosynthetics directly.