Expressing cloned genes in host cells to get their protein products requires careful selection of the host organism and some engineering on the vector DNA.
In some cases, scientists clone a gene into a host cell, like bacteria, to express it, or make it produce its protein product in large amounts. These proteins can be either used in research (e.g. to identify its function) or as a pharmaceutical ( e.g. insulin). Whole process requires meticulous planning with a guidance by the gene of interest.
One of the issues is the choice of the host organism. As they grow fast and require easier culture conditions, bacteria is the first choice. However, bacteria do not have RNA-splicing machinery. Hence, eukaryotic genes, which have large introns (i.e. noncoding regions), should be cloned as devoid of their introns if the purpose is expression. This becomes possible by using cDNA of the target gene. cDNA is a complementary DNA to mRNA. As splicing mechanisms removes introns from mRNA, cDNA carries only exons (i.e. coding regions) of a gene.
Bacteria also lacks post-translational modification mechanisms (e.g. glycosylation) that are crucial for the functioning of certain complex eukaryotic proteins. For such proteins, scientists mostly use yeast cells. Yeast is a eukaryotic organism, that grow with similar easiness to bacteria. Also, yeast cells have plasmid DNA which provides certain comfort for cloning genes. The complexity of modifications certain mammal proteins require may not be even for yeasts. In those cases, insect or mammalian cell lines come into help.
Another issue is the design of the expression vector to achieve a high expression of the protein. For this, upstream of the gene should have a promoter that is highly active in the host organism.
• Expressing cloned gene aims to produce the protein product of the gene in a high amount.
• One of the issues is which host organism to use for cloning and expressing the gene.
• After choosing a host organism, proper engineering of coding and regulatory sequences in vector DNA is necessary so that host expression machinery produces a protein in the correct form and sufficient amounts.
mRNA: An RNA molecule that is complementary to one strand of DNA and a product of the transcription process.
plasmid: A circular DNA in bacteria and protozoa that is separate from the chromosomes.
vector: Carrier DNA molecule that is used to store, amplify, express a certain gene, or any DNA sequence of interest.
promoter: A DNA sequence that is upstream of a gene and regulates its expression.
upstream: Denoting the relative position of a DNA sequence with reference to a gene, upstream means “towards 5’ end of DNA from the start site of a gene”.
RNA-splicing: An enzymatic mechanism in eukaryotic cells that removes non-coding sequences from mRNA.
coding region: A region of a gene that codes for a protein.
post-translational modifications: Attachment of chemical groups onto certain amino acids on proteins in cells
introns: non-coding sections of RNA or DNA
eukaryotic: an organism with complex cells, or a single cell with a complex structures. In these cells the genetic material is organized into chromosomes in the cell nucleus
cDNA: DNA synthesized from a single-stranded RNA by the enzyme reverse transcriptase