Practical GK-14

Antibody engineering

Aims

• To show examples of engineered proteins based on antibodies.

Objectives

After this practical you will:

• have seen examples of engineered antibodies;
• be familiar with the structure of immunoglobulin domains.

Exercise

You might find useful information in the PDB file headers, and/or in the papers describing the structures (PubMed entry for paper describing 1MOE; PubMed entry for paper describing 1NMC). For schematic representations of antibody VL and VH domains, see Figure 1.3 in Huihua Li's thesis.

(I suggest you look at questions 1(a) and 2(a) first.)

1. Protein Data Bank entry 1MOE contains the structure of an engineered diabody.

   1. Identify the chains and domains in this structure. Locate the linkers in each chain. What are the sequences of these? Comment on the residue types in the linker regions.

   2. (I recommend that you skip this part of the question.)

      The program in /chalmers/users/kemp/TDA506/practical14/vhview draws a schematic representation of the structure of an antibody VH domain. You can write out a printable encapsulated Postscript of this picture to the file 'temp.ps' in your current working directory (option "Postscript to 'temp.ps'" in the "File" menu).

      By looking at the structure and/or the PDB file, identify all of the residues in the structural framework of one of the VH domains in the structure 1MOE, and write the one-letter codes for these in the circles of the schematic diagram (either by hand or using the vhview program). Thus, find the sequences of the structural complementarity determining regions (CDRs) H1, H2 and H3.

      Write a description of how you did this (Which residue did you identify first? Which ones did you identify next? In which order did you identify residues as you navigated around the structure? Did you use RasMol to identify all of the residues, or did you identify residues at some consecutive positions simply by looking at the PDB file?).

2. Protein Data Bank entry 1NMC contains the structure of a complex between a single-chain Fv (scFv) and influenza virus neuraminidase.

   1. What is the sequence of the linker between the VH and VL domains in this scFv? The coordinates of the linker are not given in the structure file. With reference to the 3-D structure, approximately where would you expect to find linker? What is the distance between the pair of residues that will be joined by the linker?

   2. Highlight the CDR regions in the scFv. Which CDRs are in contact with the virus protein? Describe how you have interpreted "in contact with".