Cloning and Protein Expression | Thermo Fisher Scientific

There are many options available for protein expression from cloned DNA. These include cell-free extracts (in vitro expression systems), and bacterial, yeast, insect, and mammalian cell systems, each with its own advantages and drawbacks. Requirements for protein solubility, functionality, and yield are often the most important factors to consider when choosing an expression system, but the time and labor required for subcloning and optimizing expression can also have a significant impact.

Use of recombinant proteins varies widely - from functional studies in vivo to large-scale protein production for structural studies and therapeutics. Using the best expression system for your protein and application is key to your success.

The following table highlights the characteristics of the commonly-used expression hosts.

Select the best expression host for your research

Host organism	Typical applications	Advantages	Potential Challenges
Cell-free	Rapid expression screening Expression of toxic proteins Incorporation of unnatural labels or amino acids Functional assays Protein interaction studies	Rapid expression directly from plasmid Open system—easily add components to enhance protein solubility or functionality Simple format Scalable Compatible with Gateway cloning	Difficult to obtain protein yields >3 mg
Bacteria	Structural analysis Antibody generation Functional assays Protein interaction studies	Scalable Low cost Simple culture conditions Compatible with Gateway cloning	Protein solubility Minimal post-translational modifications possible May be difficult to express functional mammalian proteins
Yeast	Structural analysis Antibody generation Functional assays Protein interaction studies	Eukaryotic protein processing Scalable, with very high yields possible using fermentation (g/L) Simple media requirements	Fermentation required for very high yields Growth conditions may require optimization
Insect	Functional assays Structural analysis Antibody generation	Post-translational modifications similar to mammalian systems Higher yield than mammalian systems Compatible with Gateway cloning	Relatively demanding culture conditions
Mammalian	Functional assays Protein interaction studies Antibody generation	Highest level of correct post-translational modifications possible Highest probability of obtaining fully functional human proteins Compatible with Gateway cloning	Multimilligram per liter yields only possible in suspension cultures Relatively demanding culture conditions

Protein Expression with Gateway technology

Gateway technology allows highly efficient transfer of the gene of interest into any of a number of expression vectors from a single entry clone. The variety of expression choices available makes the Gateway system of recombination cloning ideal for protein expression studies.

The Gateway recombination cloning system is designed for highly efficient transfer of your DNA into any Gateway expression vector from a single entry clone. The extremely wide variety of Gateway cloning–compatible expression vectors available makes the Gateway system of recombination cloning ideal for protein expression studies.

One hour, room temperature cloning reactions with up to >99% efficiency deliver the clone you need
Maintain orientation and reading frame without using restriction enzymes or ligation to make expression-ready clones
Use the same clone from target identification to validation to help ensure consistent results throughout your experiment, without re-sequencing
Easily shuttle insert DNA from one expression vector to another—get flexibility while simplifying your cloning workflow
Learn more about Gateway cloning

For T7-regulated E. coli expression systems, MagicMedia Expression Medium can increase protein yields up to 10-fold. The exclusive MagicMedia formulation enables E. coli to reach culture densities 3–10-fold higher than those achieved with traditional LB + IPTG media.

Learn more about MagicMedia medium

For Research Use Only. Not for use in diagnostic procedures.