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

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

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

Purification of Fusion Proteins by Immunoaffinity Chromatographic Purification

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

Phage Display

Bacterial Display

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

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

Unidirectional Tandem Gene Array

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

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)

* 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.)

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