Question

Describe plasmid vectors and procedures used for high-level inducible expression and affinity purification of recombinant proteins using E.coli.

Answer 1

Plasmids are naturally occuring circular ,extrsomal,autonomously replicating DNA, present in many prokaryotic and few eukaryotic organisms.

Plasmids range in size from approximately 1kb to over 300 kb.

plasmids are often described as being either relaxed or stringent on the basis of the number of copies of the plasmid that are mainted within cell .relaxed plasmids are mainted at multiple copies per cell ,while stringent plasmids are present at a single copy ,or a low number of copies per cell.

plasmids provide a simple way of cloning small DNA fragments in bacterial cells

ideal plasmid vector must have small size ,high copy number, own origin of replication, restriction sites for many restriction enzymes and selectable markers

An example of widely used cloned vector is pBR322,which replicates in E.coli

Procedures used for high level inducible expression in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production.

The gene was cloned into PET-28a expression vector and transformed into E. coli BL21 (DE3).

Induction at lower temperature, lower IPTG concentrations and richer growth media during expression resulted in increased expression level.

The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay.

In addition, several attempts were made to produce soluble product and all resulted in insoluble product.

The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis.

Among the solubilization buffers examined, buffer with 1% N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solubilization and recovery.

The denaturant was removed by filtration and dialysis.