Abstract
Solubility of recombinant proteins (i.e., the extent of soluble versus insoluble expression in heterogeneous hosts) is the first checkpoint criterion for determining recombinant protein quality. However, even soluble proteins often fail to represent functional activity because of the involvement of non-functional, misfolded, soluble aggregates, which compromise recombinant protein quality. Therefore, screening of solubility and folding competence is crucial for improving the quality of recombinant proteins, especially for therapeutic applications. The issue is often highlighted especially in bacterial recombinant hosts, since bacterial cytoplasm does not provide an optimal environment for the folding of target proteins of mammalian origin. Antibody fragments, such as single-chain variable fragment (scFv), single-chain antibody (scAb), and fragment antigen binding (Fab), have been utilized for numerous applications such as diagnostics, research reagents, or therapeutics. Antibody fragments can be efficiently expressed in microorganisms so that they offer several advantages for diagnostic applications such as low cost and high yield. However, scFv and scAb fragments have generally lower stability to thermal stress than full-length antibodies, necessitating a judicious combination of designer antibodies, and bacterial hosts harnessed with robust chaperone function. In this review, we discuss efforts on not only the production of antibodies or antibody fragments in microorganisms but also scFv stabilization via (i) directed evolution of variants with increased stability using display systems, (ii) stabilization of the interface between variable regions of heavy (V (H) ) and light (V (L) ) chains through the introduction of a non-native covalent bond between the two chains, (iii) rational engineering of V (H) -V (L) pair, based on the structure, and (iv) computational approaches. We also review recent advances in stability design, increase in avidity by multimerization, and maintaining the functional competence of chimeric proteins prompted by various types of chaperones.