Useful Reminders/Links

Requirements:

1. Antibody amino acid (or DNA) sequence is available.
2. Antigen (for activating system) is commercially available at a reasonable cost.
3. Antigens should not significantly affect our mammalian cells other than their involvement in activating our system (for example, avoid EGFR or TNF-alpha as antigens).  Additionally, antigens shouldn't present significant health dangers to us (for example, anthrax spores).
4. Antigen-antibody interaction should be well-characterized.

Leader sequences

Summary Table

Chicken Lysozyme

Chicken lysozyme derives from chicken egg white.  Breaks down bacterial cell walls.

mAb name:  HyHEL-10

Sequence:  this paper

Binding interactions: this paper (interacts with discontinuous parts of peptide)

Peptide sequence: UniProt

Cross-checked peptide sequence with antibody binding interaction sites.  None of those sites have known variants/mutations.  However, peptide numbering is different on UniProt and in binding interactions paper (18aa difference).

Peptide suppliers:

Sigma-Aldrich:  1g/$49.50

Merck-Millipore:  1g/$39

HIV1 gp120 (HIV envelope glycoprotein)

gp120 is a glycoprotein derived from gp160.  gp160 is processed into gp120 and gp41, which together form a complex that binds to CD4, permitting the fusion of viral and cell membranes.

gp120 antibodies review

Peptide suppliers:

Full-length gp120 peptides are not readily available.  Check the manufacturing details to ensure that the epitope is included in the peptide.  Also check that peptide comes glycosylated.

HXB2 strain:

Abcam:  10ug/$228

MyBioSource:  50ug/$365

YU2 strain:

MyBioSource

0.5beta

mAb database:  HIV Molecular Immunology 2002 (same as this)

mAb name:  0.5beta

Sequence:  IMGT

Epitope (recognition sequence):

IMGT:  RKSIRIQRGPGRAFVTIG

HIV molecular immunology database:  RGPGRAFVTIG

**PROBLEM:  Commercially available HXB2 gp120 peptides have the amino acid sequence RKRIRIQRGPGRAFVTIG

Commercially available peptides are based on this sequence data:  NCBI, UniProt

Serine residue does contribute to binding:  0.5beta binding interactions.pdf

Tried testing binding in silico:

Performed in silico mutation using Swiss PDB Viewer:  S234R, where 234 is PDB residue number (contained in peptide P) corresponding to S residue in RKSIRIQRGPGRAFVTIG

Energy minimization with Python code from 20.320 using PyRosetta:

Looking more closely at PDB file SPV didn't add hydrogens with the mutation  - how to fix this?

VRC01

Really cool paper about gp120 single-chain antibodies in E. coli

Everything you ever wanted to know about this antibody:

Antibody identification paper (and supplementary materials, which includes sequences)

• Supplementary materials includes VRC01 variable regions amino acid sequences
• Supplementary materials includes HXB2 strain gp120 core reference sequences (Fig. S1B).  Only includes core parts of the gp120 amino acid sequence separated by GAG linkers.   One mutation S334A relative to env HXB2 sequences of commercially available peptdies. 
• Has numbers about binding interactions (including Kd!)
  • SPR
  • ITC
  • Competition ELISA

Binding information

• Includes alanine scanning table

Structural analysis

• Most natural resistance a result of variation in V5 region