Protein Protocols Protein Protocols

1036 Kipriyanov
Fig. 1. Schematic representation of the domain structure of an immunoglobulin G (A) and
recombinant single-chain antibody fragments (B). Antibody variable domains (V H, V L), peptide
linkers (L), and antigen binding sites (Ag) of Fv modules are indicated.
obtaining functional antibody fragments is to imitate the situation in the eukaryotic cell
for secreting a correctly folded antibody. In E. coli, the secretion machinery directs
proteins carrying a specific signal sequence to the periplasm (12). The SCA fragments
are usually correctly processed in the periplasm, contain intramolecular disulfide bonds,
and are soluble. However, the high-level expression of a recombinant protein with a
bacterial signal peptide in E. coli often results in the accumulation of insoluble antibody
fragments after transport to the periplasm (13,14).
It is now recognized that aggregation in vivo is not a function of the solubility and
stability of the native state of the protein, but of those of its folding intermediates in
their particular environment (15,16). The degree of successful folding of antibody fragments
in the bacterial periplasm appears to depend to a large extent on the primary
sequence of the variable domains (17,18). The overexpression of some enzymes of
the E. coli folding machinery such as cytoplasmic chaperonins GroES/L, periplasmic
disulfide-isomerase DSbA, as well as periplasmic peptidylprolyl cis,trans-isomerases
(PPIase) PpiA and SurA did not increase the yield of soluble antibody fragments
(19–21). In contrast, the coexpression of either bacterial periplasmic protein Skp/OmpH
or PPIase FkpA increased the functional yield of both phage-displayed and secreted
scFv fragments (21,22). Modifications in bacterial growth and induction conditions
can also increase the proportion of correctly folded soluble SCA. For example, lower-