Approach

Protein Expression

There is no prior way to determine which expression system will work for a new protein. The approach that the Protein Expression Core Facility (PECF) uses when starting a new project is to examine many different expression constructs simultaneously in parallel. To achieve this goal, the PECF has implemented the Gateway® expression system from Invitrogen. This system allows the PECF to generate vectors for recombinant protein expression using E. coli, baculovirus/insect cell, stable insect cell, and stable mammalian cell rapidly with no sub-cloning. Gene migration into the expression vector is mediated by the lambda recombination system. The PECF consults with the investigator's group to determine the end goal of the project and how to best design the entry clone to achieve this goal. The design of the entry clone will determine if the protein will be expressed with or without n/c terminal tags and if the tags can be removed after purification. All entry clones are prepared and sequenced by the investigator's group prior to being worked on by the PECF. The use of these tags can greatly affect the amount, solubility, and purification strategy used to isolate the expressed proteins.

Once the expression vectors are prepared, the use of different cell lines can greatly affect protein expression and solubility. The PECF primarily uses Rosetta2™ (DE3) pLacI cells for checking expression in E. coli, SF9 cells for expression using baculovirus, and COS, HEK293, or CHO cells for expression in mammalian cell lines. Rosetta2™ cells are used to remove the problem of codon usage/bias when expression heterologous proteins in E. coli, the CodonPlus® cell lines also could be used to fulfill this function. Several other cell lines are available with other unique properties, for instance: Rosetta-gami™ cells to help with folding problems associated with disulfide formation, and C43 (DE3)™ cells for membrane or membrane-associated proteins.  Temperature can also greatly affect protein expression/folding in E. coli. All E. coli expression checks are performed at 30° C and 18° C.

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How the Process Works

1. Answer questionnaire as completely as possible.
2. When the project request form is processed, the PECF will contact you via e-mail and set up a meeting to discuss your needs in greater detail.

Gateway Vectors and Flows

Slide 1: (http://www.niehs.nih.gov/research/atniehs/core/pe/approach/slide1.cfm) Using PCR to make the entry clone results in the greatest number of options when deciding how to express the protein. The design of the PCR product will dictate if the desired gene can be expressed with n or c terminal tags, or no tags at all. It is also the only way to introduce proteolytic cut sites in most of the expression vectors. The entry clone can also be made by traditional sub cloning and TOPO® cloning methods.

Slide 2: (http://www.niehs.nih.gov/research/atniehs/core/pe/approach/slide2.cfm) The generation of each expression vector is fast (~2 hrs) and can be done in parallel. Once the Entry clone is made, there are no traditional sub cloning steps in making expression vectors. This removes all problems that may arise due to conflicts with restriction sites naturally occurring within the gene. The gene is always in frame with all tags on all the expression vectors.

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