High-throughput antibody generation and characterization

A phage display library has been constructed containing over 1010 human antibodies, allowing the large-scale generation of antibodies. Over 38,000 recombinant antibodies against 292 antigens were selected, screened and sequenced, and 4,400 resultant unique clones characterized further.

Peptides selected to bind to hepatitis B virus (HBV) core protein block interaction with the long viral surface antigen (L-HBsAg) in vitro. High resolution electron cryomicroscopy showed that one such peptide binds at the tips of the spikes of the core protein shell. The peptides contain two basic residues; changing either of two acidic residues at the spike tip to an alanine greatly reduced the binding affinity. Transfection of hepatoma cells with a replication-competent HBV plasmid gave significantly reduced production of virus in the presence of peptide, in a dose-dependent manner. These experiments show that the interaction of L-HBsAg with core particles is critical for HBV assembly, and give proof of principle for its disruption in vivo by small molecules.

BackgroundIn the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.ResultsSeveral protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.ConclusionsBy analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.

As an example for studies of contacts involved in complex biological systems, peptide ligands that bind to the core antigen of hepatitis B virus (HBcAg) have been selected from a random hexapeptide library displayed on filamentous phage. Affinity-purified phage bearing aa sequence LLGRMK, or some related sequences, bound fulllength or truncated HBcAg but did not bind denatured HBcAg. The long (L), but not the short (S), hepatitis B virus envelope polypeptide, when synthesized in an in vitro system, bound firmly to HBcAg, indicating that interaction between HBcAg and the pre-S region of the L polypeptide is critical for virus morphogenesis. This interaction was inhibited by peptide ALLGRMKG, suggesting that this and related small molecules may inhibit viral assembly.(12) and is also by far the most abundant form in the 22-nm particles of HBsAg that are found in great excess over the virus in the plasma of infected individuals.The HBV core antigen (HBcAg) can be synthesized efficiently in Escherichia coli (13,14), where it assembles to form 27-nm particles equivalent morphologically to those found in the liver of infected individuals (15). This provides a convenient source of HBcAg and derivatives of it for identification of peptide sequences carried by fusion phage that bind the antigen. The L and S HBsAg. forms were synthesized in cell-free systems for analysis of their interaction with HBcAg and its inhibition by antibodies and peptides identified via the fusion phage libraries.Coliphage fd carrying random hexapeptide sequences in their gpIII protein have emerged as powerful tools for studies of ligand binding in the absence of structural or even sequence information (1-3). These phage have found use in a range of studies-including mapping the binding sites of antibodies (1, 3, 4), a chaperone protein (5), and cell-surface receptors (6).As a test system for the identification of contact regions between the components of complex biological structures such as organelles, viruses, or other multicomponent assemblies, we have used such fusion phage to explore interactions between the core and surface antigen components of hepatitis B virus (HBV). The results offer a guide to a critical stage of viral morphogenesis and approaches to its inhibition by small molecules, derived from short peptides, resembling the contact regions.HBV consists of a nucleocapsid, the small 3.2-kb DNA genome, and the viral polymerase enclosed by the core antigen of the virus, surrounded in turn by the HBV viral surface antigen (HBsAg). The viral envelope contains three different, but related, HBsAg polypeptides, which overlap extensively from their carboxyl termini and arise from variable use of initiation triplets at different points within a continuous open reading frame. The long polypeptide (L polypeptide) is the product of the entire reading frame and comprises the pre-Sl domain of 108 amino acids (or 119, depending on virus subtype) at its amino terminus followed by the pre-S2 domain of 55 amino acids and the short polypeptide...