Expression vectors

DEFINITION

• The expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for protein expression in cellsThe expression vector is a plasmid engineered to introduce a particular gene into the target cell.

• Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a portable translation initiation sequence.

• Expression vectors are used for molecular biology techniques such as site-directed mutagenesis.

Introduction

• 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 ribosomal complexes.

• 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, and in extension, proteins. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin, which is important for the treatment of diabetes.

• 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.

• Expression vectors are used for molecular biology techniques such as site-directed mutagenesis. Cloning vectors, which are very similar to expression vectors, involve the same process of introducing a new gene into a plasmid, but the plasmid is then added into bacteria for replication purposes. In general, DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein.

• Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

• Expression vectors produce proteins through the transcription of the vector's insert followed by translation of the mRNA produced, they therefore require more components than the simpler transcription-only vectors. Expression in different host organism would require different elements, although they share similar requirements, for example a promoter for initiation of transcription, a ribosomal binding site for translation initiation, and termination signals.

• Prokaryotes expression vector• Promoter - commonly used inducible promoters are promoters derived

from lac operon and the T7 promoter. A stronger promoter; Trp/Tryptophan Operon and Tac Promoter, a hybrid collection of both the Trp and Lac Operon promoters.

• Ribosome Binding Site (RBS) Follows the promoter, and promotes efficient translation of the protein of interest.

• Translation initiation site - Shine-Dalgarno sequence enclosed in the RBS, 8 base-pairs upstream of the AUG start codon.

• The basic requirement for any expression system is a promoter cloning site(s) next to it and transcriptional terminator.

• Origin suitable for replication initiation in a particular bacteria or any host.

• A selection marker gene such as antibiotic resistance gene.

• Regulator elements.

• Shine Delgarno sequence.

pLac-Z expression vectors

• Lac –Z promoter operator is in frame with lac-Z alpha fragment (the NH3 terminal part of Galactosidase gene.

• Multiple cloning sites are found in the border of NH3 end including ATG sequence.

• The presence of such restriction site sequences should not disturb the functional activity of the protein, which complements with the omega fragment of the Lac-Z produced by the bacterial cell as the complement.

• If any gene is placed in proper frame in the MCS the protein expressed will be is fused form.

• The expression of the gene can be regulated by IPTG (Isopropyl thio b-Galactoside).

pET Expression Vector:

• The size of the vector is 5700bp• It has T7 promoter adjacent to lac operator.• Next to it is a sequence called Shine Delgarno

sequence.• Adjacent to S/D sequence there are few cloning sites

such as Nde I, Nhe I and BamH I, at the end of which is T7 phage transcriptional terminator is found.

• For the expression of this gene the bacterial cell should provide T7 RNA polymerase, which is under the control of Lac-Z operator/promoter.

• The repressor produced by the bacterial lac-IQ can be regulated by IPTG.