1. Chapter 2. Molecular Biotechnology Biological System

2. Prokaryotic and Eukaryotic Organisms
Prokaryotic cells
Prokaryote (pro; before, karyon: kernel or nucleus)
No nuclear membrane
Small (0.2-2 m), mostly single-celled organisms
Eubacteria : common bacteria, e.g. E.coli, blue-green algae
Archaea (Archaebacteria)
Eukaryotic cells
Eukaryote (well-formed nucleus)
Nuclear and internal membranes  organelles
Larger than prokaryotes ( m)
Single-celled: yeast, green algae, amoebae
Multicellular: fungi, plant, animal

3. Prokaryotic cell
Eukaryotic cell

4. Escherichia coli One of the most studied organisms
Gram-negative, nonpathogenic, motile, rod shaped
Naturally found in the intestines of humans
About 22 min doubling time in rich medium at 37oC
Facultatively anaerobic
Both aerobic and anaerobic growth
Useful for recombinant protein production
Aeration is important for productivity
Other microorganisms for molecular biotechnology applications
Bacillus, Corynebacterium, Streptomyces, Pseudomonas, Zymomonas etc.

5. Gram negative vs. Gram positive
Bacterial Cell Wall
Gram Gram +

6. Divided into two groups by gram stain.
Gram-negative cell (e.g. E. coli)
Outer membrane
Peptidoglycan
Cytoplasmic (inner) membrane
Periplasm
Between inner and outer membranes
Gram-positive cell (e.g. Bacillus subtilis)
No outer membrane
Teichoic acid
Cytoplasmic membrane

7. Saccharomyces cerevisiae
Eukaryotic version of E. coli
Nonpathogenic, single-celled (5 mm) yeast
Basic research
Eukaryotic model system
e.g. cell cycle regulation, disease model
The first eukaryotic genome sequenced (1996)
Yeast artificial chromosome
Biotechnology
Production of ethanol and CO2
Alcoholic beverages and bread
Production of recombinant proteins from eukaryotic organisms
Provide protein modification
Other yeast for protein production
Pichia pastoris, Hansenula polymorpha
Schizosaccharomyces pombe, Yarrowia lipoltica
S. cerevisiae
S. pombe

8. Eucaryotic Cells Fungi Algae Protozoa Plant cells Animal cells
Yeasts --- single small cells of 5- to 10-mm size
Molds --- filamentous fungi, have a mycelial structure
Algae
Unicelluar algae (microalgae) to 30 mm
Plantlike multicelluar algae
Protozoa
Unicellular, motile, relatively large ( mm) --- amoeba
Plant cells
Animal cells

9. Secretion Pathways Function of secretory proteins
Nutrient acquisition
Cell-to-cell communication
Protection
Structure of outer surface of cell membrane
N-terminal amino acid sequence determines secretion
Signal peptide, signal sequence, leader sequence, leader peptide
Membrane barrier of prokaryotes
Gram negative bacteria
Inner membrane, periplasmic space, outer membrane
Gram positive bacteria
Single membrane
Eukaryotic secretory proteins
Modifications of secretory proteins during secretion
Glycosylation, acetylation, sulfation, posphorylation

10. Secretion in Gram-positive Bacteria
Sec complex (secretion complex)
Membrane-bound assembly of proteins
Facilitates the secretion of protein through a channel
Direct contact with the signal peptide or
Signal peptide
 binds with signal recognition complex (SRP, signal recognition particle)
 binds with SRP receptor
 contacts with Sec complex
Signal peptidase
Removal of signal peptide
Releasing the protein
Crossing the porous cell wall and proper folding

11. Secretion in Gram-positive Bacteria

12. Secretion in Gram-negative bacteria
Sec-dependent :General secretion pathway (GSP)
Passage through inner membrane
SecB + signal peptide-containing protein  SecA  Sec complex
Removal of signal peptide after transport and folding in periplasm
Passage through outer membrane
Autotransporter pathway
A region of the protein generates channel structure and removed after transport of the other part
Single accessory pathway
Transport across a single outer membrane channel
Chaperone/usher pathway
Proteins that form P pili on the surface of the cell
Type II secretion pathway (for most secreted proteins)
Gsp complex spans the periplasmic space

13. Type II Secretion Pathway in Gram-negative Bacteria

14. Secretion in Gram-negative bacteria
Sec-independent :
Type I and Type III Secretion Pathway
Each has its own protein complex that extends from the inner to the outer membrane.

15. Protein Secretion in Eukaryotic Cell
The signal sequence of a secretory protein is bound by a signal recognition particle (SRP) during protein synthesis.
The SRP attaches to a receptor on the ER membrane.
The secretory protein is translocated into the lumen of the ER.
It is folded and packaged in a transport vesicle.
It enters the Golgi network at the cis face..
A plasma membrane-specific vesicle is formed at the trans face of the Golgi network.
The secretory transport vesicle fuses with the plasma membrane and releases the protein.

16. Protein Secretion in Eukaryotic Cell

17. Protein Secretion ER lumen Golgi apparatus Secretory vesicle
Protein folding
Glycosylation
Golgi apparatus
Budding off from ER and fusion with cis Golgi
Trans Golgi
Posttranslational modification
Secretory vesicle
Fusion with the membrane

18. Eukaryotic Cell Culture
Primary cell culture
Isolation of a small sample of tissue
Treatment with protease to release cells from extracellular matrix
Growth in medium containing nutrients and growth factors
Monolayer growth
Passage or subculture after confluent growth
Limited cell division: 50~ 100 cell generations
Useful to study biochemical features of various tissues
Established cell lines
Indefinite growth by genetic changes (chromosomal change)
Useful for maintaining virus and protein expression