Basic architecture of expression vectors
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The document discusses expression vectors, which are DNA molecules that introduce specific genes into host cells for protein production, detailing their types (plasmid and viral) and essential components. Key elements include promoters, origins of replication, and purification tags, which aid in gene expression and protein isolation. Additionally, it outlines features such as start and stop codons, fusion proteins, and regulatory mechanisms to optimize protein production.
Basic architecture of expression vectors
- 1. Expression Vectors The expression vector is a DNA molecule that carries a specific gene into a host cell and uses the cell's protein synthesis machinery to produce the protein encoded by the gene. Expression vectors may be plasmid or viral. Plasmid expression vectors are used for high level expression of proteins. Viral expression vectors are useful in studying gene under endogenous promoter, in normal conditions. The expression vector must contain elements essential for gene expression. The essential elements include: strong or normal promoter, a correct translation initiation sequence such as a ribosomal binding site and an initiation codon, a termination codon, and a transcription termination sequence. Additionally, to facilitate protein purification after protein production, expression vectors usually have a purification tag, which is added to the protein sequence of interest. Figure: Basic Architecture of an expression vector
- 2. Components of an expression vector a) Selectable marker: In the absence of selective pressure plasmids are lost from the host. 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. b) 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 can be prevented by the constitutive expression of a repressor protein. c) Origin of replication: The origin of replication controls the plasmid copy number. d) Promoter: The promoter 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. e) Transcription terminator: The transcription terminator reduces unwanted transcription and increases plasmid and mRNA stability. f) 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.
- 3. To avoid formation of secondary structures (which reduces expression levels) this region should be rich in A residues. g) Start codon: Start codon is 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. h) 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. i) Protease cleavage site: Protease cleavage sites are often added to be able to remove a tag or fusion partner from the fusion protein after expression. However, cleavage is rarely complete and often additional purification steps are required. j) Multiple cloning site: A series of unique restriction sites that enables to clone gene of interest into the vector. k) 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.
- 4. Table: Components of expression vector Goal Component 1. Insert cargo into the plasmid and verify the insert sequence accuracy • MCS – restriction sites OR recombination regions • 5’ and 3’ Primer sites for sequence verification 2. Insert plasmid into cells, enable the plasmid to replicate inside the host & select for cells carrying the plasmid • Backbone compatible with cloning method • Origin of replication • Selection marker and/or screening marker 3. Transcribe mRNA from the plasmid • Promoter (constitutive or inducible) operator, terminator 4. Translate mRNA into protein • Ribosome Binding Site, start codon, stop codon 5. Promote proper folding of nascent protein • co-expression of chaperones • Solubilization tags • custom-designed synthetic RBS • Codon-optimized ORF 6. Detect or Purify target protein • Epitope tags (His) • reporters (GFP)