抗体噬菌体展示

Periplasmic Stress Response Systems-two Systems
• Stress: heat shock, hyperosmotic shock, expression of outer membrane proteins defective in folding, pH etc.
• The pIII protein domain N1 is required during infection for the translocation of the DNA into the cytoplasm and the insertion of the coat proteins into the membrane.
• Cpx pathway, regulate DsbA, RotA, and PpiD, DegP, CpxP
• It is composed of CpxA and CpxR.
Phage-display Vectors
Phage Display Vectors-M13KE, Wild-type
• Infection is a multistep process requiring interactions with the F conjugative pilus and the bacterial TolQ, R, and A cytoplasmic membrane proteins.
• Heat shock response-σ32, inducing production of proteins, control of PpiD catalyst
• σE pathway, control the synthesis of at least 11 proteins, such as DegP, FkpA • RseA, ResB are negative regulators of σE
Antibody Phage Display
20180803 Meiling Xiong
Filamentous Phage Biology
• Major coat protein, gene VIII protein, pVIII, 50 amin-acid
• 5 molecules of 406-residue gene III, pIII,
• DsbA, DsbG catalyze the formation of disulfide bonds • DsbC catalyzes the rearrangement and isomerization of incorrectly formed disulfide bonds. • Peptidyl-prolyl isomerase (PPI): catalyze the interconversion between the cis and trans form of the peptide bond X-pro. • RotA, FkpA, SurA, PpiD. • Other proteins: Skp • Degradation: DegP, Protease III, Prc, OmpT (proteases)
• IG region, intergenic region, do not encode proteins; site of origin for DNA synthesis. Packaging signal.
Filamentous Phage Display- The Phage Life Cycle
• Group2: gene VII, IX, VIII, III, VI encode capsid proteins
• Group3: gene I, XI, and IV encode proteins that are involved in the membrane-associated assembly of the bacteriophage.
Protein Folding And Degradation In The Periplasm
• The periplasm has an oxidizing environment to allow formation of specific disulfide bonds that aid in both the folding and stabilization of the mature protein.
• pVIII proteins are arranged in an overlapping shingle-type array with a symmetry.
• The pVIII molecules are packed quite tightly, as only the outside 3 residues are accessible to digestion by proteases.
• The infectivity of the phage is destroyed by deleting from the phage either the N1 or N1 and N2 domains of the gIII.
• A peptide or protein is fused N-terminally to some or all copies of the CT domain, or N2-CT domain.
• The cytoplasm has a reducing environment. • The oxidation, reduction, and isomerization of disulfide bonds in
proteins are accomplished by the enzymatic systems. • Thiol-disulfide oxidoreductase
• The infectivity of the phage is restored by adding the N1 or the N1-N2 complex. They are fused or chemically coupled a ligand, which binds to the phage display peptide or protein.
• The other end of the particle has about 5 molecules each of the small hydrophobic pVII and pIX proteins. This end contains the packaging signal (PS) and is the first part of the phage to be assembled. Only pIX is exposed, pVII is buried.
• N2, is responsible for binding to the F pilus.
• CT is essential for forming a stable phage particle.
Filamentous Phage Display- The Phage Genome
• Group1: gene II, V, X encode proteins required for the replication of the phage genome.
• Two routes for infectivity restoration: in vivo and in vitro.
In vivo
In vitro
Protein Translocation Across The Cytoplasmic Membrane
• The periplasm is a gel-like compartment, with diffusion coefficients 100-dold lower thant those in the cytoplasm.
• For translocation, four mechanisms have been identified in E.coli. Three need other proteins, one does not.
• signal or leader sequeroteins • The Sec system • The Tat system • SRP system • Spontaneous insertion pathway
Filamentous Phage Display-the Assembly Process
• This process requires the five capsid proteins, the three assembly proteins, ATP, a proton motive force, and at least one bacterial protein, thioredoxin硫氧 还蛋白.
• Substrate: pV-DNA complex • Process: initiation, elongation, termination
Filamentous Phage Display-selectively Infective Phage
• SIP exploits the modular structure of the gIII protein.
• F pilus is required for the conjugal transfer of the F plasmid DNA from a donor cell into a recipient bacterium lacking the plasmid.
Filamentous Phage Display- Replication And Protein Synthesis