Re: Cloning and introns

Hi Audra,

I have not heard of introns affecting the stability of the final protein,
unless it is the case that you have missed an intron and the expressed
protein has become balderdash. As you might know, bacteria – because I’m
assuming you express the protein in E. coli – don’t use introns: these are
eukaryotic novelties.

Aggregation of over-expressed protein is not uncommon at all. There can be
many reasons for this. First and simplest, you express a much larger amount
of protein than what the cell can support. Another reason is that, although
you might have corrected the coding of the protein from eukaryotic to
prokaryotic codon usage, the proteins that chaperone the correct folding of
the protein have trouble handling the ‘intruder’. Thirdly, organisms vary
in the use of so-called targeting sequences: parts of the newly translated
protein show structures that tell the cell that it should be sent into the
nucleus, or locked into to endoplasmic reticulum, or sent to the
mitochondria, or exported etc. If proteins end up in the wrong place, they
can/will aggregate. If the amounts of protein are normal, then it will be
cut into pieces and recycled, but as stated above, cloned proteins are
usually massively expressed.

You are right in suspecting that introns do have a function: they are not,
as previously labelled, junk DNA. Not all of it at least. But their role is
in regulating the transcription (of the DNA into RNA), either so that a
given gene is more or less expressed, or actually making variants of
proteins, controlling the connection of different exons (the ‘opposite’ of
introns). The near infinite variation of antibodies is generated in this
way. The spacing of genes in eukaryotes has also been shown to be
tragically important: remember the French patients that received gene
therapy and ended up with cancer some years ago? The introduced gene had
nested in the wrong place within the so-called junk DNA, triggering the
onset of leukaemia.

There has been evidence that part of the ‘junk DNA’ is expressed in some
way, maybe making RNA with enzymatic activity, either on it’s own, or as
part of still unknown protein/RNA complexes (you know: as in ribosomes for
instance). This is really a very exiting new line of study!

I hope I have managed to answer your question.
Kind regards,