Question

If your goal is to cause a specific protein to be expressed transiently via an episomally maintained gene, could you just exclude all introns and the exons not associated with that transcript variant when choosing what DNA to package into your plasmid for that gene? So far as I know, the role of introns is usually in allowing alternative splicing and in modifying gene expression. But if you're only interested in one transcript variant, you wouldn't need alternative splicing to occur on the episome, and gene expression is handled in plasmid design by the selected promoters and enhancers.

For those reasons, could the introns (and exons not used for that transcript variant) be excluded? And would that affect the protein produced?

Answer 1

Excluding introns and non-essential exons from the DNA sequence for plasmid-based gene expression is a common practice when specific protein expression is desired, as introns are non-coding and exon sequences must be accurately joined. Introns may play regulatory roles, but in transient expression systems and non-eukaryotic hosts, their removal may have minimal impact on the desired protein production.

Step-by-step explanation:

When considering the expression of a specific protein via an episomally maintained gene, introns and non-essential exons could indeed be excluded from the DNA sequence that is cloned into a plasmid. Since introns are non-coding sequences that are removed from the pre-mRNA during RNA splicing, they do not contribute to the amino acid sequence of the encoded protein. In a eukaryotic system, introns can have regulatory functions and might be involved in alternative splicing, which allows a single gene to produce multiple protein isoforms. However, if the goal is to produce a specific transcript variant, then only the exons that are part of this variant need to be included in the plasmid design, along with regulatory sequences such as promoters and enhancers to control expression.

It is crucial to consider that exon sequences need to be joined in the correct reading frame to produce a functional protein. Any errors in splicing out introns could lead to a frameshift and a nonfunctional protein. By constructing a plasmid without introns, the concern of splicing errors is eliminated, which can be particularly beneficial for heterologous expression in a system such as bacteria that does not have the splicing machinery present in eukaryotic cells. However, the removal of introns may impact aspects of gene expression and mRNA processing, although in a transient expression system, these impacts may be minimal or non-essential.