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 some HXB2 strain gp120 core reference sequences (Fig. S1B).  It seems that this includes only parts of the gp120 amino acid sequence separated by GAG linkers.  (Why?)
• 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