There has been a recent surge in demand for recombinant proteins across the medical, research and biotechnological realms. However, this is no surprise because, in the past, the only primary way to obtain a specific protein was first to separate it from a natural source. This was not only inefficient but was pretty time-consuming.
Thanks to advances in recombinant biological molecular practices, it is now possible for researchers and medical personnel to clone the DNA of a particular protein in an expression vector. Selecting the right vector is critical to the success of the operation in question. This is why we put together a list of key factors you should consider when selecting a vector for your molecular biology experiment.
The Purpose of the Vector
One of the key factors to consider while choosing a recombinant protein vector is what you intend to do with it. For instance, is it cloning or expression? Wondering what the difference is? Well, cloning vectors are the way to go if you are trying to generate many copies of your gene. This is because they have features used in the insertion and removal of DNA fragments into a gene and have multiple cloning sites.
Expression vectors, on the other hand, are useful if you are trying to generate the gene into mRNA and protein. This is primarily because they contain organism-specific sequences such as RBS sequences and promoters, among many others.
The Vector Size
This is a must-consider factor, whether you are cloning or working with an expression vector. Luckily, the primary element to consider when it comes to the vector size is whether you are working with a small or large DNA fragment.
It is the rule of the thumb to keep the vectors small for the convenience of manipulation. For instance, most plasmids can efficiently cope with inserts that are up to 15kb. Anything bigger than that may alter the replication process and result in stability issues.
So what do you do if you have a larger DNA insert? Well, there are vectors specifically designed for such applications. But even these can only go up to 52kb and have a different origin of replication. Also, it is vital to note that the bigger the vector, the lower its transfection efficiency and its stability.
The Selectable Marker
A selectable marker refers to an antibiotic resistance gene. These markers offer the host cells containing the recombinant vectors the traits they require to survive artificial selection. In respect to that, you are required to choose a selectable marker for both expression and cloning vectors, as this is the only way to identify a positive transformant. The kind of selectable marker to use is mostly dependent on the host cell type in use. In that light, the two major types of markers are drug-resistant markers and auxotrophic markers.
Drug-resistant markers often involve the use of a gene encoding enzyme that, in turn, inactivates a particular antibiotic. Auxotrophic markers, on the other hand, allow the cells to survive in the medium even without the vital nutrients.
In cloning, you can either use a positive selection, which means only cells with the selectable marker will survive or, a negative one, which means only cells that lack the marker survive. In expression, the option of the selectable marker mostly depends on the host cell type in use.
You must choose the right vector as it directly influences the quality of the recombinant proteins. Or you can save yourself the stress of selecting vectors by purchasing ready to use recombinant proteins from reputable brands such as ImmunoDX.