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 (VH) and light (VL) chains through the introduction of a non-native covalent bond between the two chains, (iii) rational engineering of VH-VL 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.
Highlights
Antibodies are widely used for medical applications such as disease diagnosis and therapy (Grilo and Mantalaris, 2019)
The single-chain variable fragment, a rational polypeptide design, consisting only of variable regions from heavy (VH) and light (VL) chains, joined together by a linker, maintains antigen binding capacity (Humphreys, 2003; Andersen and Reilly, 2004) and can be produced in prokaryotes, such as Escherichia coli (E. coli) or Brevibacillus choshinensis (B. chosinensis) (Hu et al, 2017), along with high yield, which keeps the cost of production low (Power and Hudson, 2000; Terpe, 2006; Rosano and Ceccarelli, 2014; Gupta and Shukla, 2017)
Despite the advantages of scFvs, they have a few drawbacks that limit their therapeutic potential, such as (i) deteriorated stability because of their propensity to readily aggregate under thermal stress (Jager and Pluckthun, 1999a; Demarest and Glaser, 2008); (ii) a short serum half-life of
Summary
Antibodies are widely used for medical applications such as disease diagnosis and therapy (Grilo and Mantalaris, 2019). Robin Curtis’s group at the University of Manchester investigated the aggregation propensity of argininerich scFv under denaturing condition: substitution of arginine residues in scFv with lysine significantly reduced aggregation (Austerberry et al, 2019) This diverse knowledge of protein nature in scFv may assist engineers with consensus-based design of antibody fragment for generating stabilizing mutations to pre-existing scFvs (Steipe, 2004) or bispecific antibody fragments (Jordan et al, 2009). Andreas Plückthun’s group at University of Zurich designed a stabilized scFv from human VH germline sequences by analyzing hydrophobic core, pairing of hydrogen bonds, clusters of charge, and packing of β-sheets, leading to reduction of G = 20.9 kJ/mol as well as improvement of scFv expression yield by 4-fold (Ewert et al, 2003) They could stabilize scFv by CDR grafting to more stable framework, using a structure-based analysis (Ewert et al, 2004). Georgiou and Ellington at the University of Texas at Austin used the Rosetta modeling program (Sircar et al, 2009; AdolfBryfogle et al, 2018) to predict amino acid substitutions for anti-HA33 scFv stabilization and confirmed a melting temperature increase of 4.5◦C by antigen-binding enzyme-linked immunosorbent assay (ELISA) after thermal stress for 2 h at 70◦C (Lee et al, 2019)
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