Expression vectors

General considerations for expression vectors

1. General Considerations for Expression Vectors, Promoter and Other elements. By : Kanchan Rawat M.Sc. Biotechnology Jamia Hamdard University

2. Expression Vectors These are the plasmids that carry cargo (insert DNA) into cells and allow the cargo DNA to be efficiently expressed. 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 . Expression vectors have promoters with on/off switches because too much production of foreign gene can be toxic. One commonly used promoter is a mutant version of the lac promoter, lacUV , which drives a very high level of transcription, but only under induced conditions.

3. Expression Vectors Have Tightly Regulated Promoters: eg lacUV promoter. To stimulate transcription, the artificial inducer, IPTG, is added. IPTG binds to the LacI repressor protein, which then detaches from the DNA. This allows RNA polymerase to transcribe the gene. Before IPTG is added, the LacI repressor prevents expression of the cloned gene.

4. Expression Vectors components 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 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)

5. Polylinkers : Every vector contains a defined number of recognition sites for restriction enzymes that cut the vector sequence only once to enable the cloning of a DNA fragment. These sites usually lie closely together and these sections are referred to as polylinkers (or multiple cloning sites, MCSs). These regions comprise 50–100 bp on average and may contain up to 25 restriction sites for single-cutting restriction enzymes. Promoter: In order to transcribe a DNA fragment in bacteria, a promoter is needed that ensures reliable and strong mRNA synthesis with RNA polymerase. The strength of the promoter does not only depend on the interaction with RNA polymerase but also modulated by interaction with protein eg . CAP protein of lac promoter.

6. The strongest promoters are found in bacteriophages are T5 or T7 promoter. In prokaryotes, promoter specificity of an RNA polymerase molecule is mediated by sigma factor. There are also hybrid promoters composed from various bacterial promoters. The ptac promoter, for example, is a hybrid of the promoter of the lacZ gene, which can be induced by the addition of isopropyl-b-d- thiogalactoside (IPTG) and the promoter of the tryptophan operon .

7. Commercially available yeast expression vectors can contain constitutively active or inducible promoters. Constitutive promoters include, for example, the GAP promoter of the gene for glyceraldehyde-3-phosphate dehydrogenase. Examples for inducible promoters are: ( 1) the AOX1 promoter of the alcohol oxidase gene, which is induced by methanol and is suitable for protein expression in Pichia pastoris , ( 2) the galactose -inducible promoters Gal1 and Gal10 for protein expression in Saccharomyces cerevisiae ( 3) thiamine-inducible promoters nmt1, nmt42, and nmt81 for protein expression in Schizosaccharomyces pombe .

8. Promoters in Eukaryotic Expression Vectors for Mammalian Cells : In order to express proteins in eukaryotes, a promoter must be located in front of the cloned cDNA to enable its transcription in the cellular system. Viral promoters are frequently used, as these ensure strong constitutive expression. The most often used promoters are the CMV promoter derived from the cytomegalovirus and the SV40 promoter of the simian virus 40.

9. Ribosome binding sites: Initiation of translation requires a ribosome binding site. In prokaryotes the efficiency of translation is affected by primary and secondary structure of mRNAs in the region of 30s ribosome subunit. SD sequences with initiation codons AUG or GUG form RBS. SD sequence is 4-9 bp long and is positioned. Kozak sequence: In eukaryotic expression vectors should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA .

10. Polyadenylation tail: In eukaryotes polyadenylation tail at the end of the transcribed pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: stabilizes mRNA production. polyadenylation of the primary transcript seems to be a prerequisite for the formation of translatable mRNA. The process involves: cleaving off the end of the transcript and attaching the poly(A) sequence. Several components are needed: a nucleolytic enzyme complex and poly(A) polymerase.

11. Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors. Termination Sequence: B acterial expression vectors carry specific sequences that enable them to form stable mRNA secondary structures after transcription. These prevent RNA polymerase from continuing the synthesizing process beyond this site. Without transcription terminators, a whole vector sequence would be transcribed into one long mRNA in a runaway transcription .

12. A termination of the transcript also enhances the stability of mRNA. Some transcription terminators consist of partly viral (e.g., phage lambda) and partly bacterial termination sites, while others are derived from exclusively viral sequences (e.g., T7 bacteriophage ). many bacterial expression vectors also carry translation termination sites. Often, only fragments of genes are cloned into vectors without their sequence-specific stop codons . A short TG-rich sequence before the transcription terminator acts as a stop codon in each of the possible reading frames.

13. Fusion Sequence: In prokaryotes, the purification process of the recombinant protein through affinity chromatography can be facilitated by the expression of a fusion protein This is why many vectors already contain sequences leading to the expression of N- or C-terminal peptide sequences (tags ). In contrast to the fusion components of prokaryotic expression vectors, which are very cost-effective in affinity purification, for the short peptide tags in many eukaryotic expression vectors, the most important criterion is their antigenicity. The most frequently used tags are the c- Myc tag, the hemagglutinin (HA) tag, and the FLAG tag.

14. References Winnacker , from genes to clones Genscript.inc Wink, Introduction to molecular biotechnology