James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
Protein expression, a requisite task for any biomedical scientist, can be applied to numerous laboratory methods. However, expressing a protein inside a cell can take months to perfect and is fraught with pitfalls. Even when optimal in vivo expression conditions are optimized, the protein may be degraded inside the cell. Or worse, the wrong protein may be expressed.
However, there is a silver lining in this cloudy picture: Proteins can be expressed outside the cell. Expressing a protein outside the cell in a laboratory can also be tedious, but it is a necessary evil for anyone interested in studying the protein without growing liters of cells and enduring hours of chromatographic protein purification. There are many benefits and drawbacks to using a eukaryotic, mammalian, cell-free system to express a protein in vitro; some will be discussed in this article.
A multitude of applications can be performed using a cell-free, in vitro protein-expression system. Applications include genetic verification and detection, functional protein analysis, molecular interaction detection, molecular structure and localization analyses, molecular diagnostics, high-throughput screening and functional genomics.
Setting up
A typical setup of an in vitro protein can be simple or complex, depending on the application. However, there are a few common elements that go into every reaction. First, you need to obtain the open reading frame for the protein you want to express. One way you can obtain this is by using PCR to amplify the DNA from a cellular, viral or a plasmid source. The DNA source must also contain the proper cis-acting transcription elements: promoters, enhancers, repressor-binding sites and high-yield translation factors, such as IRES and Kozak consensus sequences.
The cis-acting elements may be native or cloned into the DNA source to flank the open reading frame. These elements ensure optimal messenger RNA (mRNA) transcription initiation and termination, mRNA stability and translation initiation. Cell-free, in vitro protein-expression kits typically contain one or more of these cis-acting elements in their expression vector (plasmid).
You also will need the necessary trans-acting factors to transcribe, and later translate, your favorite open reading frame. These include not only general and cell-specific transcription factors necessary to bind to those oh-so-important cis-acting elements in your source DNA, but also RNA-stabilizing factors, translation factors and ribosomes. The latter are necessary for proper translation of the protein. Cell-free, in vitro protein-expression kits come with these factors in the form of a cell lysate from, typically, human or rabbit cells.
Common obstacles
A typical reaction time for cell-free, in vitro protein expression is two hours. Although reaction time is a major benefit with these systems, there are plenty of roadblocks to their use. One commonly cited in research papers is the fact that cell-free expression systems typically do not yield more than 3 mg of the protein of interest. Some applications, such as high-throughput screening, require a higher yield, so it is necessary to optimize the sequences in the cis-acting elements of your expression vectors to avoid the low-yield problem.
Ways to optimize the cis-acting sequences include adding a highly active enhancer/promoter region, like that in Cytomegalovirus (CMV) immediate early genes; stabilizing mRNAs by adding coding sequence for attachment of a polyadenosine tail; and adding a Kozak consensus sequence or Internal Ribosomal Entry Site (IRES) to the mRNA to optimize its translation into protein. Because each kit comes with a different expression vector, it is essential to become aware of the cis-acting elements in your kit’s vector.
Expressing a protein in a cell-free system has many benefits. However, like other life-science laboratory methods, it has its fair share of drawbacks. Being aware of the necessary cis-acting elements and trans-acting factors contained in your kit is crucial to successful protein expression.
The image at the top of the page is New England Biolabs' pSNAPf Vector.