Melvyn Little

Bio: Melvyn Little is an academic researcher from German Cancer Research Center. The author has contributed to research in topics: Antigen & Antibody. The author has an hindex of 50, co-authored 150 publications receiving 7453 citations. Previous affiliations of Melvyn Little include University of Texas at San Antonio & University of Texas Health Science Center at San Antonio.

Complete amino acid sequence of beta-tubulin from porcine brain

Abstract: The primary structure of porcine brain beta-tubulin was determined by automated and manual Edman degradation of six sets of overlapping peptides. The protein consists of 445 amino acid residues and has a minimum of six positions that are heterogeneous, indicating at least two beta-tubulins in porcine brain. Comparison of the optimally aligned sequences of alpha-tubulin and beta-tubulin indicates that 41% of their primary structures are identical. A region rich in glycyl residues is similar both in sequence and predicted secondary structure to the phosphate binding loop of several nucleotide binding enzymes. beta-Tubulin contains a highly acidic COOH-terminal region that resembles the NH2-terminus of troponin T.

Targeting recombinant antibodies to the surface of Escherichia coli: fusion to a peptidoglycan associated lipoprotein.

Abstract: To target recombinant antibodies to the surface of Escherichia coli, we have fused single-chain variable domains to its peptidoglycan associated lipoprotein (PAL). The fusion protein was able to bind antigen and was tightly bound to the murein layer of the cell envelope. Antibody-PAL had little effect on cell growth and viability. In contrast, the expression of single chain antibody alone eventually resulted in cell lysis. Immunofluorescence studies on unfixed cells showed that functional antibodies were accessible at the surface of intact bacteria. This could provide a means of isolating single cells producing specific antibodies from libraries in E. coli by fluorescence assisted cell sorting (FACS). Pal fusions may also be of general interest for the presentation of proteins at the surface of E. coli as, for example, in the production of live vaccines.

Complete amino acid sequence of alpha-tubulin from porcine brain.

Abstract: The amino acid sequence of alpha-tubulin from porcine brain was determined by automated and manual Edman degradation of eight sets of overlapping peptides. It comprises 450 residues plus a COOH-terminal tyrosine that is present only in 15% of the material. A region of 40 residues at the COOH-terminus is highly acidic, mainly due to 16 glutamyl residues. This high concentration of negative charge suggests a region for binding cations. At least six positions, most of them around position 270, are occupied by two amino acid residues each. Several of these exchange sites were assigned to specific peptides by analysis of the purified corresponding fragments. These data indicate four alpha-tubulins in porcine brain. Although alpha-tubulin on the whole is unrelated to other proteins, there are regions that can be correlated to sequences of the myosin head, to actin, to tropomyosin, and to troponins C and T.

Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.

Abstract: The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits.

Directed evolution of novel binding proteins

Abstract: In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Methods for producing members of specific binding pairs

Abstract: A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

Engineered antibody fragments and the rise of single domains

Abstract: With 18 monoclonal antibody (mAb) products currently on the market and more than 100 in clinical trials, it is clear that engineered antibodies have come of age as biopharmaceuticals. In fact, by 2008, engineered antibodies are predicted to account for >30% of all revenues in the biotechnology market. Smaller recombinant antibody fragments (for example, classic monovalent antibody fragments (Fab, scFv)) and engineered variants (diabodies, triabodies, minibodies and single-domain antibodies) are now emerging as credible alternatives. These fragments retain the targeting specificity of whole mAbs but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Antibody fragments have been forged into multivalent and multispecific reagents, linked to therapeutic payloads (such as radionuclides, toxins, enzymes, liposomes and viruses) and engineered for enhanced therapeutic efficacy. Recently, single antibody domains have been engineered and selected as targeting reagents against hitherto immunosilent cavities in enzymes, receptors and infectious agents. Single-domain antibodies are anticipated to significantly expand the repertoire of antibody-based reagents against the vast range of novel biomarkers being discovered through proteomics. As this review aims to show, there is tremendous potential for all antibody fragments either as robust diagnostic reagents (for example in biosensors), or as nonimmunogenic in vivo biopharmaceuticals with superior biodistribution and blood clearance properties.

Making Antibodies by Phage Display Technology

Abstract: Antibody fragments of predetermined binding specificity have recently been constructed from repertoires of antibody V genes, bypassing hybridoma technology and even immunization. The V gene repertoires are harvested from populations of lymphocytes, or assembled in vitro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Rare phage are selected from the repertoire by binding to antigen; soluble antibody fragments are expressed from infected bacteria; and the affinity of binding of selected antibodies is improved by mutation. The process mimics immune selection, and antibodies with many different binding specificities have been isolated from the same phage repertoire. Thus human antibody fragments have been isolated with specificities against both foreign and self antigens, including haptens, carbohydrates, secreted and cell surface proteins, viral coat proteins, and intracellular antigens from the lumen of the endoplasmic reticulum and ...