1. Fusion Proteins Fusion protein Cleavage of fusion proteins
to protect the cloned gene product from attack by host cell proteases
need a proper reading frame
Cleavage of fusion proteins
Linkage of two proteins with nonbacterial protease recognition sequence
Ile-Glu-Gly-Arg : C-terminal end cleavage by factor Xa

2. Uses of Fusion Proteins
For secretion (ompF) and quantifying (lacZ)
(Figure 6.9)
For efficient purification
(Table 6.3, Figure 6.10, Figure 6.11)
His tail (6-10 aa): Ni2+ binding, elution by imidazole
Strep-tag (10 aa): Streptavidin binding, eluction by iminobiotin
MBP (40 kDa): Amylose binding, elution by maltose
GST (25 kDa): Glutathione binding, elution by reducing agent
Flag (8 aa): Flag Ab binding, elution by low calcium

4. Mab against the marker peptide is immobilized on a solid support and acts as a ligand to bind the fusion protein.

5. Purification of Fusion Proteins by Immunoaffinity Chromatographic Purification

6. Surface Display
Display proteins as fusion proteins with a surface proteins (filament protein, pilus protein)
Phage display (Fig. 6.12)
Protein pIII of M13 phage
Bacterial display (Fig. 6.13)
Bacterial outer surface protein and a foreign target protein
E. coli OmpA, OmpF : outer membrane protein
E. coli PAL: peptidoglycan-associated lipoprotein
Target protein in N or C terminus, or in the middle

7. Phage Display

8. Bacterial Display

9. Usage of Surface Display
By this method, cDNA libraries can be screened for clones that carry specific coding sequence.
Overexpression of peptides or proteins
Expression of antigenic determinant of the parasite Plasmodium falciparum , causative agent of malaria, by inserting into surface-exposed loops of the major outer membrane protein F (OprF) from Pseudomonas aeruginosa
Possible usage as vacccines

10. Unidirectional Tandem Gene Array
Multiple copies of the gene of interest into a low-copy-number plasmid can be used more effective than one copy into a high-copy-number plasmid.
Because high copy plasmid will be a burden.
Generation of tandem gene array
Using complementary 3-nucleotide extensions

11. Unidirectional Tandem Gene Array

12. Unidirectional Tandem Gene Array
TypeIIS restriction enzyme
Cut random sequence outside of the recognition site
Formation of different three-nucleotide 5’ extensions
Use Pfu (Pyrococus furiosus) polymerase for PCR
Unlike Taq polymerase, it does not add an extra base to the ends of the PCR products

13. Translation Expression Vectors
Ribosome binding site
Shine-Dalgarno sequence
5’AGGAGGU3’ (mRNA)
6-8 bases
Strong ribosome binding site
Location of ribosome binding
usually 4-7 bases ahead of the initiation codon (AUG)
8 bases ahead in pKK233-2 (Figure 6.17)

14. * start codon at mRNA level: AUG
* start codon at DNA level: ATG (coding strand)
(Although the template for transcription is the complementary stand (TAC), the strand containing ‘TAC’ is called noncoding strand.)

15. Translation Expression Vectors
The first few codons of the gene of interest must not contain nucleotide sequences that after transcription can fold back, i.e., form intrastrand loops. (Figure 6.16)
Note that G can also base pair to some extent with U.
When a cloned gene has codons that are rarely used by the host cell
use other host cell (eukaryotic host cell)
codon usage (for codon optimization)
use a host cell engineered to overexpress several rare tRNAs (Figure 6.18)
Strain is commercially available