Marc D. Better

Role of the Xis protein of bacteriophage λ in a specific reactive complex at the attR prophage attachment site

Abstract Phage λ controls its integration and excision by differential catalysis of the forward and reverse reactions. The λ Int protein is required for both directions, but Xis for excision only. Previous electron microscopic observations have shown that Int protein forms a stable, condensed protein-DNA complex with the phage ( att P) and prophage left ( att L) substrate sites, but not with the host ( att B) or prophage right ( att R) sites. We have found that Int and Xis together produce a stable, condensed complex with att R. The att R complex involves the P region DNA to the left of the crossover point (O site). In contrast, the att P complex includes DNA on both sides of the crossover point (P and P′), and the att L structure involves the P′ DNA to the right of O. In the presence of Int and Xis, the att L and att R sites form a paired structure. We conclude that the role of Xis is to provide a distinct reactive structure at att R, allowing att L and att R to pair efficiently.

Chimeric mouse-human anti-carcinoma antibodies that mediate different anti-tumor cell biological activities

Two chimeric mouse-human antibodies, ING-1 (IgG1, kappa) and ING-2 (IgG1, lambda), have been constructed starting from anticarcinoma mouse hybridomas. These antibodies bind to different tumor-associated antigens which are present on human breast carcinoma cell lines at 10(5)-10(6) antigens/cell; ING-1 binds to a 40-kD membrane glycoprotein, while ING-2 binds to a glycoprotein of greater than 300 kD. In competitive binding experiments, both chimeric antibodies have identical binding activity to the parental mouse antibodies. The antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytolysis (CDC) activities of these antibodies were studied on carcinoma target cell lines. ING-1 mediates potent ADCC, but ING-2 had undetectable or very weak ADCC activity. ING-2 ADCC activity was significantly reduced by the addition of human serum, but ING-1 ADCC was unaffected. Neither ING-1 nor ING-2 mediated CDC of breast carcinoma cell lines, but ING-1 mediated CDC of a colon carcinoma cell line. ING-1 antibody-antigen complexes are stable on the target cell surface for at least 2 hours, while much of bound ING-2 is lost from the surface of cells due to internalization or shedding. The activities of these antibodies confirm that the target antigen plays an important role in the biological effector functions triggered by cell-surface-bound antibodies. Both of these chimeric antibodies are candidates for further study as immunoconjugates for cancer diagnosis or therapy, and the unconjugated ING-1 antibody has promise for cancer therapy due to its potent activation of ADCC.

Production of antifungal recombinant peptides in Escherichia coli.

Antifungal peptides derived from the human bactericidal/permeability-increasing protein (BPI) were produced in Escherichia coli as fusion proteins with human BoneD. Bacterial cultures transformed with the gene encoding the fusion protein were grown to a high cell density (OD(600)>100), and induced with L-arabinose to initiate product expression. Fusion protein accumulated into cytoplasmic inclusion bodies and recombinant peptide was released from BoneD by acid hydrolysis at an engineered aspartyl-prolyl dipeptide linker. Acid hydrolysis of purified inclusion bodies at pH <2.6 followed Arrhenius kinetics and did not require prior inclusion body solubilization in detergents or denaturants. Surprisingly, at pH <2.6 and 85 degrees C, cell lysis and aspartyl-prolyl hydrolysis with concomitant peptide release occurred simultaneously. Bacterial cultures were, therefore, adjusted to approximately pH 2.6 with HCl directly in the bioreactor and incubated at elevated temperature. Peptide, which is soluble in the aqueous acidic environment, was separated from the insoluble material and purified using column separation techniques. Recombinant peptide was separated from the hydrolyzed bioreactor culture with >76% recovery and a final peptide purity of >97%. Antifungal peptide prepared by recombinant and solid phase synthesis methods demonstrated similar activity against Candida sp. in a broth microdilution assay.

Chimeric immunoglobulin light chains are secreted at different levels : influence of framework-1 amino acids

Immunoglobulin light chain proteins are generally thought to be readily secreted without their corresponding heavy chains; non-secreted light chains have been viewed as aberrant forms. We have re-examined this assumption by expressing chimeric mouse-human light chains constructed for 12 mouse antibodies (mouse variable regions fused to a human kappa light chain constant region) in Sp2/0 and CHO cells. Five of the 12 light chains were either poorly secreted or not secreted at all. There was approximately a five-fold difference in the levels of secreted light chain between the highest poor secretor and the lowest good secretor. All of these light chains formed functional chimeric IgGs, which were secreted at similar levels, when co-expressed with their respective chimeric mouse-human heavy chains (mouse variable regions fused to a human gamma-1 heavy chain constant region). The influence of variable region amino acids on light chain secretion was examined by replacing the Framework-1 region of three poorly-secreted chimeric light chains with that of a readily-secreted light chain. For two of the light chains, secretion levels increased approximately 30- and 100-fold relative to that of the unmodified light chains. Comparison of the Framework-I amino acid sequence of the poorly- and readily-secreted light chains revealed an asparagine (N) and proline (P) at positions 11 and 12, respectively of these poorly-secreted light chains and a leucine (L) and serine (S) in the same region for some of the readily secreted light chains. Alteration of the NP to LS for one of the poorly-secreted light chains resulted in an approximately seven-fold increase in light chain secretion over that of the native form of the poorly-secreted light chain. We conclude from these studies that poor secretion can be a naturally occurring state for normal light chains and that amino acids within Framework-1 contribute to poor secretion for some of the light chains.