ABSTRACT
OBJECTIVE: Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation. RESULTS: Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10. CONCLUSION: Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.
Subject(s)
Mycobacterium tuberculosis/enzymology , Pichia/growth & development , Serine Proteases/genetics , Serine Proteases/metabolism , Aldehyde Oxidase/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromatography, Affinity , Fungal Proteins/genetics , Gene Expression Regulation/drug effects , Homologous Recombination , Humans , Interleukin-10/metabolism , Monocytes/cytology , Monocytes/drug effects , Monocytes/immunology , Mycobacterium tuberculosis/genetics , Pichia/genetics , Pichia/metabolism , Protein Domains , Protein Engineering , Serine Proteases/chemistry , Serine Proteases/pharmacology , THP-1 Cells , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Technology is the basis of scientific progress and is an essential component for continued competitiveness in industry. The development of a new drug candidate is a long and expensive process, in which a molecule undergoes several stages of research (both pre-clinical and clinical) before being approved for commercialization. Scientific progress has revolutionized the pharmaceutical industry and reshaped the processes by which new drugs are discovered, investigated, and developed. Currently, the influence of genomic variations in drug metabolism must be better understood to predict an individual´s response to a given treatment. Employing genomics tools, an individual's genetic profile may be obtained and used as the basis for prescription of the best treatment option, thus personalizing medicine. In this review, we discuss how current mainstream genomic technologies used in clinical pharmacology research can accelerate the identification of populations that can benefit the most while reducing adverse events.
Subject(s)
Biomedical Research/methods , Genomics/methods , Pharmacology, Clinical/methods , Biomedical Technology/methods , Drug Design , Drug Discovery/methods , Humans , Precision Medicine/methodsABSTRACT
Immunotherapy is defined as the use of the immune system or components of it, such as key immune molecules, to fight diseases or invading infectious agents. Modern biotechnology provides industrial versions of immune molecules (components of the immune system) naturally produced by the human body. Immune molecules such as monoclonal antibodies are used as therapeutics in several disease conditions. In recent years a new group of antibody based molecules has been developed to replace monoclonal antibodies, given their ability to overcome some of the limitations of the latter. The first clinical trials with these new molecules have been very encouraging and the promise is that they will be released to the market very soon. This in turn has stimulated more research on new versions of antibody based therapeutics by biotechnological companies supported by the pharmaceutical industry and in many cases in collaboration with academic institutions.
Subject(s)
Antibodies/therapeutic use , Biotechnology/methods , Immunotherapy/methods , Animals , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/therapeutic use , Drug Industry , Humans , Immunologic Factors/therapeutic useABSTRACT
Genomic variation largely explains the differences in an individual's response to drug treatments. A field of genomic medicine focuses on the identification of genetic polymorphisms and gene mutations involved in the development and progression of disease. Another part focuses on the development of genetic tests to accompany medical prescriptions, to predict how certain patients respond to therapy with a given pharmacological agent. The field of predicting responses to drugs has different strategies and methods, among which we find: the use of liver microsomes, cell models, monitoring of probe drugs, assays with recombinant proteins and recently the use of microarray platforms or DNAchips.
