Terminator cloning vectors

Fig. 7.11 Schematic diagram of foreign gene expression in plant. P promoter, T terminator, pGA482 plant cloning vector, Km kanamycin resistance gene, Tc tetracycline resistance gene.

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. 

PelZ is a hydrophilic protein of 420 amino acids with a short hydrophobic sequence at its N-terminal end which has Ae characteristics of the signal sequences of exported proteins. The signal peptide may be 24 amino acids long, which would corroborate wiA the usual length encountered in prokaryotes. The molecular cloning of the pelZ gene in an expression vector pT7-6 allowed for the specific 35S-cysteine-methionine raAo-labelling of PelZ in E. coli K38. We could detect, in crude extracts, the presence of a precursor and a mature form of PelZ. After cell fractionation, Ae mature form of PelZ could be localized in Ae periplasm of E. coli. So PelZ appears to be a protein exported by Ae Sec-dependent system of translocation. 

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