반응형
Cell surface display 특징
a high throughput manner에서 directed evolution을 위한 가장 유용한 도구이다.
다양한 host cell(Gram(+) bacterial, Gram(-) bacterial, yeast, insect cells, baculorias, mammalian cells)에서 사용된다.
세포표면에 display함으로서 유전자형과 표현형을 linkage한다.
기능으로는 desiging, engineering, therapeutic, proteins, vaccines, biosensors, biocatalysts에 사용된다.
E.coli Expression system
- Well-characterized genetics
- Ease of manipulation
- Variety of strains and many research tools available
- The rapid and high density growth conditions
Two membrane이고 membrane proteins는 Display of Proteins의 좋은 target이 된다.
PECS : Periplasmic expression with cytometric screenin
1. Library of secreted proteins expressed in bacteria
2. Incubation with fluorescently tagged ligand
3. Fluorescence-activated cell sorting(FACS)
4. Isolation of fluroescent, ligand-binding cells
APEx
Protein anchoring to periplasmic side of inner membrane
- N-terminal display by first 6 aa of NIpA (CDQSSS)
- Loop grafted
- C-terminal display by fusion to gpⅢ
Periplasmic Trapped Display of FC
Pe1B leader peptide fusion without anchoring or capturing motifs
Retention of homodimeric Fc in spheroplastst at optimized conditions
Bacterial Display systems & Limitaions
|
Display Systems
|
Limitations
|
|
Outer membrane surface display
|
Difficult to display large complex protein molecules
|
|
Periplasmic space display (PECS)
|
Large binding ligands or antigens (>10kDa) are not accessible to the periplasmic space
|
|
Anchored periplasmic display (APEx)
|
Spheroplasting (low viability) PCR recovery, subcloning, and transformation befor next round sorting (3 days)
|
Surface Display on Staphylococcus Bacteria
Gram positive와 Gram negative가 존재한다.
특히, Gram positive bacteria는 다음과 같은 특징을 갖고 있다.
- Thick peptidoglycan layer
- succesful display of scFvs and affibodies(작은 항체)
- Display level of proteins should be imporved
Bacterial Surface display의 장/단점
장점
High transformation efficiency
Enables the creation of big library than yeast surface display library
Fast growth speed and screeing
Easy application for simple bacterial production
단점
Poor protein folding compared to eukaryotic hosts
No glycosylation
Display of toxic proteins → affect cell physiology → poor display of proteins and viability
