Soluble expression of m2a1 and m2a2 after panning against sp62 as tested by SDS-PAGE (left) and Western blot (right).

<p>Soluble expression of m2a1 and m2a2 is indicated by arrows.</p

Primers for limited mutagenesis of the m01s loop DE.

*<p>K  =  T + G, H  =  A + T + C, W  =  A + T, M  =  A + C.</p

Binding of m2a1 and mutants to sp62 (mut 1– replaced loop BC in m2a1 to its original sequence in m01s; mut 2– replaced loop DE in m2a1 to its original sequence in m01s; mut 3– replaced loop FG in m2a1 to its original sequence in m01s).

<p>Binding of m2a1 and mutants to sp62 (mut 1– replaced loop BC in m2a1 to its original sequence in m01s; mut 2– replaced loop DE in m2a1 to its original sequence in m01s; mut 3– replaced loop FG in m2a1 to its original sequence in m01s).</p

Size exclusion chromatography of purified m2a1.

<p>The left insert is a standard curve while the right one shows purified m2a1. Calculated M.W. according to amino acid sequence of m2a1 is 15.4 kD.</p

Comparison of CH2 loop BC and germline VH H1.

<p>The frequency of amino acids occurring in H1 according to IMGT website was calculated by WebLogo. The amino acids were colored as follows: polar G, S, T, Y, C (Y is classified as “polar” amino acids by WebLogo and excluded from the “hydrophobic” amino acids listed below although it is also hydrophobic): green; neutral Q, N: purple; basic K, R, H: blue; acidic D, E: red; hydrophobic A, V, L, I, P, W, F, M: black. The dominant length of H1 is eight amino acids (41 from 53 analyzed sequences); three sequences are nine amino acid long and nine sequences are ten amino acid long.</p

Binding of m2a1 to sp62 and neutralization of HIV-1.

<p>(A) Left: binding of m2a1 (▪) (EC₅₀ = 51 nM) and m01s (•) to sp62; binding of scrambled sp62 to m2a1 (□), m01s (○) was used as negative control. Right: binding of m66.6 to sp62 (▴) and scrambled sp62 (Δ). (B) Percentage inhibition of a panel of viruses pseudotyped with Envs of HIV-1 primary isolates by m2a1 at 5 µM (77 µg/ml) and m6 (positive control) at 0.5 µM (14 µg/ml), respectively. m01s at 5 µM (70 µg/ml) was used as negative control. Data are presented as mean ± SD.</p

Primers for rational mutagenesis of the m01s loop BC.

*<p>R  =  A + G, K  =  T + G, W  =  A + T, M  =  A + C, N  =  A + C + G + T, Y  =  C + T, H  =  A + T + C.</p

Primers for precisely grafting VH H3 onto the m01s loop FG.

*<p>D  =  A + T + G, Y  =  C + T, R  =  A + G.</p

Binding of m2a1 to shFcRn.

<p>(A) Binding of yeast-expressed m2a1 and Fc to shFcRn at pH 6.0 (red) and pH 7.4 (blue) measured by flow cytometry. Fluorescence intensity shifts were observed in both cases of m2a1 and Fc. The expression of m2a1 and Fc on yeast cell surface was detected by the mouse anti-human CH2 mAb. PE-streptavidin was used as negative control. (B) Binding of m2a1 and Fc to shFcRn tested by ELISA. The binding of m2a1 to shFcRn at pH 6.0 (▪) and pH 7.4 (□) was tested while that of Fc to shFcRn at pH 6.0 (•) and pH 7.4 (○) was used as control. m2a1 showed good pH-dependent binding to shFcRn although the EC₅₀ at pH 6.0 was lower than that of Fc to shFcRn. Data presented as mean ± SD.</p

Sequence alignment of randomly selected clones with 8 amino acid mutations in loop BC, 5 amino acid mutations in loop DE, and VH H3 grafted onto loop FG in the m01s-based library.

<p>Nineteen of twenty five clones (76%) were expressed correctly and are shown here.</p