How to choose a pertinent Expression Vector?

Introduction

If you're one among the many researchers who have solely used a single expression vector just because it’s the one you're familiar with and it's easy to create new constructs by altering the gene insert. By this article, you may come away feeling assured to tinker with the components of your vector so as to contour your workflow or to get better expression results. 

What is Expression Vectors?

The Expression vector is used in genetic engineering that permits a specific gene to be not only cloned but also expressed in a host cell. The vector is built to contain appropriate regulatory sequences, like a promoter and operator so that the host-cell machinery will transcribe the gene and translate the resultant messenger RNA to synthesize the corresponding protein. Such vectors are therefore essential for the manufacture of, for instance, mammalian proteins by bacterial host cells. Expression vectors used in prokaryotes are usually based on plasmids or phages, or plasmid–phage hybrids (phagemids).

Several vector components are necessary to attain all of the steps resulting in high-level expression of the properly folded protein, so depending on the goal of your experiment you need to make a decision whether your gene of interest to be expressed from an extra chromosomal plasmid or whether you want to integrate your gene into the genomic DNA of your host cell. If you would like to express a protein at high levels so as to purify it or if you want to try and do reporter assay, where the overexpression driven by the strong promoter is suitable. Expression from a plasmid is simple and might be ideal but in some cases, you'll have to integrate your expression cassette into the host DNA using viral vectors which can be easy and extremely efficient, however, viral-mediated gene transfer isn't targeted. Which means genomic integration may occur anywhere within the genome. 

Expression Vector components

You need to be ready to insert your cargo and so verify that it has been inserted accurately, this can be most frequently done with the assistance of restriction enzymes which recognizes specific cleavage sites, the region of the vector where you'll insert your gene of interest contains loads of different restriction enzyme recognition sites right in a row, this can be known as the multiple cloning site or MCS. There are alternative cloning strategies that require special vectors containing recombination regions or other adaptations. After you insert your cargo or anytime you acquire or modify a plasmid you'll need a simple way to verify the accuracy of the insert sequence thus most vectors embody common sequencing primer sites like t7 and m13 is flanking the MCS. Most DNA sequencing services will provide primers for those sites at no cost, whereas if you would like a custom primer it'll cost more, so you may search for 5 Prime and 3 prime sites just for your convenience and for sequence verification.

Next, you ought to insert the plasmid into cells for certain delivery strategies; you need to confirm that your plasmid backbone is compatible. For instance, For viral-mediated delivery, your backbone must contain components needed for viral packaging then you need some way for the plasmid to initiate its own replication within the host by recruiting transcriptional machinery proteins, this can be referred to as an origin of replication and you need a way to spot cells carrying the plasmid, so you don't have a mixed pool of transformed and untransformed cells, this may be accomplished with a selection marker like antibiotic resistance which will enable solely the transformed cells to survive otherwise you could use a screening marker that allows you to visually see which cells are transformed and which are not for instance, through blue-white screening.

Once you've got the cells that are carrying your plasmid you need to transcribe mRNA from the plasmid, this count upon a transcriptional promoter. Reckoning on your experiment you may opt for a constitutive promoter or an inducible promoter the promoter region includes the sequence to which polymerizes will bind and also operator regions, where enhancers or repressors may bind and of course you need to dictate where transcription ought to end by including a terminator sequence.

The next task is to translate mRNA into a protein, so like with endogenous mRNAs your expression construct must embody a ribosome binding site and a start codon to permit the ribosome to initiate translation and you also want a stop codon to end translation and to promote proper folding of a nascent protein. Not all proteins have known chaperones or single chaperones. There are many alternative approaches that may be more powerful and widely applicable. For instance, you can add a solubilization tag to your expression constructor, you can custom design a synthetic ribosome binding site, that may hamper the translation initiation rate to permit enough time for folding to occur and you should contemplate codon, optimizing your open reading frame which can adjust the rate of translation and elongation again either to hurry it up to maximize protein yield or to slow it down in order to permit enough time for proper folding of the nascent protein.

Finally, to envision your protein in a gel or western blot you'll need to add an epitope tag that is just a tiny molecule, typically a short peptide that is readily bound by an antibody and which is not expected to alter the structure or function of your target protein. Some epitope tags like HIS are particularly useful for purifying proteins, by using a column or other sort of affinity purification you can design an expression construct such that the epitope tag can easily be removed after purification. Before you persist to characterize your target protein in addition to using an epitope tag, you would possibly want to use a reporter such as a fluorescent protein-like GFP.

Conclusion 

When it involves the expression vector, the cloning vector has to be mentioned. In general, both the expression vector and the cloning vector are synthetically constructed circular DNA sequences. Typically, an expression vector is said to be more complex than a cloning vector. Accordingly, there are many different expression vectors that could be used in different expression systems. The decision of which vector to use depends on the expression system and many other factors. A pertinent expression vector will facilitate the successful expression of the target protein and, if necessary, protein purification.

References

https://www.sciencedirect.com/science/article/abs/pii/S1046592818300433

https://link.springer.com/article/10.1007/s12033-019-00201-6

https://www.thoughtco.com/gene-cloning-and-vectors-definition-and-major-types-375681

https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpet.23

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610161/