Directed Evolution of Human Heavy Chain Variable Domain (V_H) Using <i>In Vivo</i> Protein Fitness Filter

<div><p>Human immunoglobulin heavy chain variable domains (V_H) are promising scaffolds for antigen binding. However, V_H is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the V_H domain, we performed <i>in vivo</i> protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of <i>E. coli</i>. After screening a human germ-line V_H library, 95% of the V_H proteins obtained were identified as V_H3 family members; one V_H protein, MG2x1, stood out among separate clones expressing individual V_H variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in V_H. Comparison of the crystal structures of the V_H variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the V_H interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered V_H acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the V_H variant as a scaffold was qualified as most of V_H clones selected randomly were expressed as soluble form in <i>E. coli</i> regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected V_H conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.</p></div

Far-UV CD spectra for the detection of reversible folding.

<p>(A) MG2x1. (B) MG8-6. (C) MG8-14. (D) Modified MG8-14 [L50W]. Black lines indicate profiles for V_H in native state at 25°C; red lines indicate the profile for V_H denatured at 85°C; and green lines indicate profiles for V_H renatured at 25°C.</p

Soluble expression level of screened V_H domains.

<p>SDS-PAGE of soluble (A) and insoluble (B) fractions of <i>E. coli</i> expressing each V_H domains selected from the frame-mutation library of MG2x1 were loaded as follows: 1, V_HH (camel single-domain antibody); 2, HEL4; 3, MG2x1; 4, MG8-14; 5, MG2-47; 6, MG2-55; 7, MG2-57; 8, MG2-59; 9, MG4-2; 10, MG4-5; 11, MG4-6; 12, MG4-7; 13, MG4-12; 14, MG4-13; 15, MG4-17; 16, MG4-20; 17, MG4-28; 18, MG4-32; 19, MG4-33; 20, MG8-4; 21, MG8-5; 22, MG8-6; 23, MG8-8; 24, MG8-11; 25, MG8-12; 26, MG8-13. Lanes labeled ‘MW’ contained protein size markers (10 and 15 kD). U indicates fractions from a culture with no isopropylβD-1-thiogalactopyranoside.</p

Thermodynamic stability.

<p>(A) Representative V_H domains selected from the germ-line library. (B) Representative V_H domains selected from the MG2x1 frame-mutation library. The black bold line indicates the profile of the parental V_H, MG2x1, prior to mutation. Folding fraction was converted from the temperature-scouting CD profile at a fixed wavelength (230 nm).</p

Analyses of the structural features for MG2x1, MG8-4, and MG8-14.

<p>Analyses of the structural features for MG2x1, MG8-4, and MG8-14.</p

The recovery yields of selected V_H after thermal stress. Data are means and standard deviation for three independent treatment of heat denaturation within the same sample.

a<p>Absorbance at 280 nm at 25°C.</p>b<p>Absorbance at 280 nm after heating (85°C) followed by cooling (25°C). Aggregates after heating were removed by centrifugation.</p>c<p>The recovery yield was defined as the fraction of soluble V_H remaining after heating at the denaturation temperature (85°C).</p

Frequency of mutations of the randomly chosen V_H domains after screening of the combinatorial frame-mutation library of MG2x1 via TAPE.

a<p>Possible substitution of amino acids into the frame-mutation library (%) according to the given codon degeneracy of primers used for mutagenesis of MG2x1 as shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098178#pone.0098178.s002" target="_blank">Table S1</a>.</p

Humoral immune response of BALB/c mice.

<p>Six mice (per group) were received multiple intravenous injections of PBS (P), MG2x1 (A), MG2x1 plus CFA (A), MG8-14 (B), MG8-14 plus CFA (B), V_HH (C), or V_HH plus CFA (C) at week 3 (w3), week 6 (w6), and week 9 (w9). Six mice (per group) were received multiple injections of 1 µg MG8-14 intramuscularly (im, D), 1 µg MG8-14 subcutaneously (sc, D), 10 µg MG8-14 intramuscularly (im, D), 10 µg MG8-14 subcutaneously (sc, D), 1 µg V_HH intramuscularly (im, E), 1 µg V_HH subcutaneously (sc, E), 10 µg V_HH intramuscularly (im, E), or 10 µg V_HH subcutaneously (sc, E) at week 2 (w2), week 6 (w6), and week 10 (w10).</p

Structures of MG2x1, MG8-4, and MG8-14.

<p>(A) Structure of MG2x1 with CDRH1 (yellow), CDRH2 (red), and CDRH3 (blue). Mutation sites for the MG2x1 mutation library are indicated as sticks (magenta). (B) Antibody light chain, in surface rendering (cyan), is shown to highlight the relative locations of CDR H1–H3 and the mutation sites in MG2x1 (magenta). The circle indicates the cavity area. (C) Superposition of three V_H domains (MG2x1, MG8-4, and MG8-14), showing the variation in the loop in the CDRH3 region (blue).</p

SDS-PAGE of soluble and insoluble fractions of <i>E.x coli</i>.

<p>(A) Previously characterized V_H domains (V_H2, V_H3, V_H6, DP47d, and HEL4). (B) V_H domains chosen randomly from the human V_H germ-line library (RD1–3) or selected from the human germ-line library using TAPE (MG4x4-44, MG4x4-25, MG10-10, and MG2x1). Cultures expressing each V_H domain were harvested after induction at 25°C for 3.5 h, and soluble (S) and insoluble (I) fractions were prepared. Lane ‘MW’ contains a protein size marker; the size of each marker is indicated (in kD) to the left of each panel. In both panels, the mobilities of V_H domains correspond to the 15-kD protein size marker. Different parts from separating gels are grouped to align expression patterns for soluble and insoluble fraction of each V_H domain.</p