Prokaryotic Expression Systems E. coli, Bacillus subtilis, Staphylococcus carnosus, Streptomyces lividans Prokaryotic promoter—ribosome binding site—MCS— transcription termination site Prokaryotic selectable marker

Expression of cloned genes in bacteria Expression vectors contain promoter sequences (pro) that direct transcription of inserted DNA in bacteria and sequences required for binding of mRNA to bacterial ribosomes (Shine-Delgarno [SD] sequences). A eukaryotic cDNA inserted adjacent to these sequences can be efficiently expressed in E. coli, resulting in production of eukaryotic proteins in transformed bacteria.

Necessary Features of expression vector: •Origin of replication •Drug resistance marker •A promoter •Transcription terminator •Restriction sites Expression Vectors with or without specific tags…… Vectors for non-fusion proteins pKK223-3 pSVK 3 PSVL SV40 Vectors for fusion proteins pGEX pQE pET pEZZ 18

Promoters of Importance in Biotech lac trp tac (or trc) phage l pL phage T7 gene 10

Used to avoid problems with digestion of foreign proteins by proteases Fusion proteins Used to avoid problems with digestion of foreign proteins by proteases Used to aid purification of foreign proteins, often by affinity chromatography Often a rare protease cut site is added to the fusion partner Eg., For small peptides poly-arginine,Histidine tail Large peptides Maltose binding protein( MBP) Chitin binding domain Cellulose binding domain Glutathione S- transferase( GST)

Features of Host --prokaryotic systems ex: E coli Disadvantages Advantages •Many references and much experience available e.g. E. coli. •Wide choice of cloning vectors. •Gene expression easily controlled. •Easy to grow with high yields. •Product can be designed for secretion into the growth media. Low expression No post-translational modification Degradation by bacterial proteases . •Biological activity and immunogenicity may differ from natural protein. •High endotoxin content in gram negative.

Eukaryotic Expression Systems Yeast, Aspergillus niger, Baculovirus-Insect Cells, Mammalian Cells (e.g. Chinese Hamster Ovary cells) Eukaryotic promoter-MCS-transcription termination site Eukaryotic selectable marker Fig. 2

Features of eukaryotic systemsyotic : Yeast Advantages Unlike prokaryotic expression systems, allow for posttranslational modification (PTM) of proteins (e.g., glycosylation, phosphorylation, hydroxylation, disulfide bond formation, processing of propeptides) •Lacks detectable endotoxins. •Fermentation relatively inexpensive. Disadvantage Expression less easily controlled. • Glycosylation not identical to mammalian systems.

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