PET expression vectors

The recombinant system for peptide production involved designing synthetic oligonucleotides, which could be annealed, strand extended, and then cloned into the pET-SUMO vector to generate a fusion protein. This fusion protein was expressed in E. coli BL21(DE3) cells by IPTG induction and then cells lysed by freeze-thaw lysis followed by sonication. The lysate was passed... 

The recombinant C3 exoenzyme that we used is a modified enzyme that does not contain the signal peptide, but has Met-Ala attached to Ser of the mature enzyme (Kumagai et a/., 1993). The modified gene was cloned into pET 3a vector, resulting in pET 3a C3, which allows the enzyme to be expressed under control of the bacteriophage T7 promoter. E. coli strain BL21 (DE3) cells are transformed with pET 3a C3. 

Figure 3.16 Typical commercial protein expression vectors, (a) pET-24a (+) the origin of replication (on), kanamycin resistance...

There are a number of approaches for the expression of proteins in E. coli. A modern approach to protein expression is to make use of purpose designed pDNA expression vectors such as the pET vector family, freely available from commercial sources (Figure 3.16). This pDNA expression vector family comprises a T7 RNA polymerase promoter for transcription, hence... 

An expression vector, such as pET 5 plasmid, has the components of any normal cloning vector (e.g., origin of replication, selectable marker, multiple cloning site), but it also has the ability to have the inserted DNA be transcribed. It has a promoter for RNA polymerase, such as T7 polymerase, and a termination sequence. These border the multiple cloning site. These vectors are used with a cell line that makes T7 RNA polymerase when induced. 

Fig, 1, pET-derived expression vectors harboring various fusion partners, (a) Schematic representation of the expression cassette, (b) List of the vectors, fusion tags, and their targeted cellular localization. MBPmaltose-binding protein, GB1 pi -domain of the Streptococcal protein G, Trx Thioredoxin, DsbA Protein Disulfide Isomerase A, DsbC Protein Disulfide Isomerase C, GST Glutathione S-transferase, 7 l/Tobacco Etch Virus. For the DsbC and DsbA fusions, two versions were constructed, for secretion Into the periplasm and for intracellular expression. 

We chose to use the pET system G ovagen) as this has previously produced large quantities of functional proteins with other genes in our hands. Three expression vectors were constructed ... 

Vector The expression vector pET-3a/CRABP I contains the complete coding region of bovine CRABP I cloned into the plasmid pET-3A, such that the ATG start site of the CRABP I cDNA begins at an Ndel site. The CRABP I cDNA was placed downstream and under control of the T7 promotor and upstream from the transcnption terminator. 

The expression vector pET-3a/CRABP II is similar in design and function to pET-3a/CRABP I. With pET-3a/CRABP II, CRABP II may be expressed and punfied as described above for CRABP I. 

The choice of vectors obviously depends upon the expression system that will be used and there is a wide availability of optimized reagents from both commercial and academic sources. For expression in E. coli, the most widely used system for HTP protein production is the pET/T7 promoter vector developed by Studier which makes use of T7 RNA polymerase to direct expression of cloned... 

---