1. Foreign Gene Expression and Protein Production in Prokaryotes and Eukaryotes Prokaryotic Expression Systems Fusion Proteins Biofilms Secretion Eukaryotic Expression Systems Posttranslational modifications (PTMs)

2. Prokaryotic Expression Systems E. coli, Bacillus subtilis, Staphylococcus carnosus, Streptomyces lividans Prokaryotic promoter—ribosome binding site—MCS— transcription termination site Prokaryotic selectable marker Fig. 6.19

3. Potential Problems in Prokaryotic Expression Systems Low expression Degradation by bacterial proteases Improper folding Oxygen limitations Biofilm formation

4. 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

5. Protein Secretion Requires a bacterial signal peptide sequence In gram negative bacteria like E. coli, the protein is secreted to the periplasmic space, as opposed to the media in gram positive bacteria Secretion affords some degree of protection against bacterial proteases

6. Copyright © 2010 ASM Press American Society for Microbiology 1752 N St. NW, Washington, DC 20036-2904 Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten Chapter 6 Manipulation of Gene Expression in Prokaryotes Figure 6.32. Expressing a foreign protein generates a metabolic load on host bacteria.

7. 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. 7.4

8. Eukaryotic Expression Systems Unlike prokaryotic expression systems, allow for posttranslational modification (PTM) of proteins (e.g., glycosylation, phosphorylation, hydroxylation, disulfide bond formation, processing of propeptides)

9. Examples of some eukaryotic expression vector constructs Fig. 7.10 K-Kozak sequence, S-signal peptide, T-protein affinity tag, P-protease site Fig. 7.24