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Eukaryotic genes are quite complex compared to their prokaryotic counterparts. They contain distinct regions called exons and introns. Exons are sequences that code for proteins, whereas introns are non-coding sequences that must be removed. In eukaryotic cells, the process called **splicing** removes these introns to produce mature mRNA. This splicing step is crucial because only the exons are meant to be translated into proteins.
However, bacteria do not perform splicing. Without intron removal, the mRNA that a bacteria would produce from a eukaryotic gene would be non-functional and unable to guide protein synthesis accurately. This fundamental difference in gene processing is a major hurdle when expressing eukaryotic genes in bacteria.

The process of mRNA splicing is vital for eukaryotic gene expression, enabling the correct sequence of exons to be joined together after intron removal. Once the transcription of a eukaryotic gene's pre-mRNA is complete, the splicing machinery called the spliceosome cuts out the introns. This ensures that the resultant mRNA is ready to be translated into a functional protein in the ribosome.
When trying to express eukaryotic genes in bacteria, the lack of splicing equipment is a key issue. Since bacteria naturally do not remove introns, any inserted eukaryotic DNA containing these non-coding regions would result in faulty, non-functional proteins. To overcome this, scientists often need to use cDNA, which is the intron-free version of the eukaryotic gene.

Promoters are essential DNA sequences that initiate the transcription of a gene. These segments are recognized by RNA polymerase and other regulatory factors like enhancers. Eukaryotic promoters differ significantly from bacterial promoters in structure and sequence. This difference means that even if introns were removed, a eukaryotic promoter might not initiate transcription in a bacterial system.
To achieve successful gene expression in bacteria, an appropriate bacterial promoter must replace or accompany the eukaryotic sequences. Such tailored adjustments allow the bacterial RNA polymerase to recognize and start accurately transcribing the gene into mRNA. Without these changes, even correctly spliced eukaryotic genes will remain inactive in a bacterial host.

Protein folding is a critical step in ensuring that a newly synthesized protein is functional. Eukaryotic proteins often have complex structures that can be difficult to replicate in bacterial cells. Variations in codon usage between eukaryotes and bacteria can affect the abundance and availability of tRNA necessary for translation.
Additionally, the folding environment in bacterial cells is different from eukaryotic cells. Even if a eukaryotic protein is synthesized, improper folding can result in a non-functional or unstable protein. Factors like the different chaperone proteins in bacteria play a role, often requiring further engineering or aid to ensure the protein molds into its necessary shape. Sufficient folding interventions are necessary to produce desired and functional proteins within bacterial cells.