Transcript Foreign Gene Expression and Protein Production

Foreign Gene Expression and Protein
Production in Prokaryotes and Eukaryotes
•Prokaryotic Expression Systems
•Fusion Proteins
•Biofilms
•Secretion
•Eukaryotic Expression Systems
•Posttranslational modifications (PTMs)
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
Potential Problems in Prokaryotic Expression Systems
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Low expression
Degradation by bacterial proteases
Improper folding
Oxygen limitations
Biofilm formation
Fusion proteins
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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
Protein Secretion
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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
Chapter 6
Manipulation of Gene Expression in Prokaryotes
Figure 6.32. Expressing a foreign protein generates a metabolic load on host bacteria.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
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
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)
Examples of some eukaryotic
expression vector constructs
Fig. 7.10
Fig. 7.24
K-Kozak sequence, S-signal peptide, T-protein affinity tag, P-protease site