VPB 321: Animal Biotechnology (2+1)

EXPRESSION VECTORS

• An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell.
• Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery.
• The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
• The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA.
• After expression of the gene product, the purification of the protein is required; but since the vector is introduced to a host cell, the protein of interest should be purified from the proteins of the host cell. Therefore, to make the purification process easy, the cloned gene should have a tag. This tag could be histidine (His) tag or any other marker peptide. View image...

Essential features of an expression vector

1. Selectable marker. In the absence of selective pressure plasmids are lost from the host. Especially in the case of very high copy number plasmids and when plasmid-borne genes are toxic to the host or otherwise significantly reduce its growth rate. The simplest way to address this problem is to express from the same plasmid an antibiotic-resistance marker and supplement the medium with the appropriate antibiotic to kill plasmid-free cells.
2. Regulatory gene (repressor). Many promoters show leakiness in their expression i.e. gene products are expressed at low level before the addition of the inducer. This becomes a problem when the gene product is toxic for the host. This can be prevented by the constitutive expression of a repressor protein. The lac-derived promoters are especially leaky. These promoters can be controlled by the insertion of a lac-operator sequence.
3. Origin of replication. The origin of replication controls the plasmid copy number
4. Promoter. The promotor initiates transcription and is positioned 10-100 nucleotides upstream of the ribosome binding site. The ideal promoter exhibits several desirable features:
   • It is strong enough to allow product accumulation up to 50% of the total cellular protein.
   • It has a low basal expression level (i.e. it is tightly regulated to prevent product toxicity).
   • It is easy to induce.
5. Transcription terminator. The transcription terminator reduces unwanted transcription and increases plasmid and mRNA stability
6. Shine-Delgarno sequence. The Shine-Dalgarno (SD) sequence is required for translation initiation and is complementary to the 3'-end of the 16S ribosomal RNA. The efficiency of translation initiation at the start codon depends on the actual sequence. The concensus sequence is: 5'- TAAGGAGG -3'. It is positioned 4-14 nucleotides upstream the start codon with the optimal spacing being 8 nucleotides. To avoid formation of secondary structures (which reduces expression levels) this region should be rich in A residues
7. Start codon. Initiation point of translation. In E. coli the most used start codon is ATG. GTG is used in 8% of the cases. TTG and TAA are hardly used.
8. Tags and fusion proteins. N- or C-terminal fusions of heterologous proteins to short peptides (tags) or to other proteins (fusion partners) offer several potential advantages:
   • Improved expression. Fusion of the N-terminus of a heterologous protein to the C-terminus of a highly-expressed fusion partner often results in high level expression of the fusion protein.
   • Improved solubility. Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
   • Improved detection. Fusion of a protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody or a binding protein (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) allows for detection of a protein during expression and purification.
   • Improved purification. Simple purification schemes have been developed for proteins fused at either end to tags or proteins which bind specifically to affinity resins
9. Stop codon. Termination of translation. There are 3 possible stop codons but TAA is preferred because it is less prone to read-through than TAG and TGA. The efficiency of termination is increased by using 2 or 3 stop codons in series.