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: Bacterial 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