Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

A roadmap to directed enzyme evolution and screening systems for biotechnological applications

Enzymes have been long used in man-made biochemical processes, from brewing and fermentation to current industrial production of fine chemicals. The ever-growing demand for enzymes in increasingly specific applications requires tailoring naturally occurring enzymes to the non-natural conditions found in industrial processes. Relationships between enzyme sequence, structure and activity are far from understood, thus hindering the capacity to design tailored biocatalysts. In the field of protein engineering, directed enzyme evolution is a powerful algorithm to generate and identify novel and improved enzymes through iterative rounds of mutagenesis and screening applying a specific evolutive pressure. In practice, critical checkpoints in directed evolution are: selection of the starting point, generation of the mutant library, development of the screening assay and analysis of the output of the screening campaign. Each step in directed evolution can be performed using conceptually and technically different approaches, all having inherent advantages and challenges. In this article, we present and discuss in a general overview, challenges of designing and performing a directed enzyme evolution campaign, current advances in methods, as well as highlighting some examples of its applications in industrially relevant enzymes.

The next step in biology: a periodic table?

Systems biology is an approach to explain the behaviour of a system in relation to its individual components. Synthetic biology uses key hierarchical and modular concepts of systems biology to engineer novel biological systems. In my opinion the next step in biology is to use molecule-to-phenotype data using these approaches and integrate them in the form a periodic table. A periodic table in biology would provide chassis to classify, systematize and compare diversity of component properties vis-a-vis system behaviour. Using periodic table it could be possible to compute higher- level interactions from component properties. This paper examines the concept of building a bio-periodic table using protein fold as the fundamental unit.

Controlling proteolytic degradation of the methionine enriched MB-1Trp protein

Protein design is currently used for the creation of new proteins with desirable traits, which include a superior nutritional value. One of the challenges of protein design in this area is to achieve the production of stable native-like proteins that resist the proteolytic pressure of the organism used for its production (the bioreactor). We report here the identification of a specific peptide bond sensitive to E. coli proteolysis in the designer protein MB-1Trp. In an attempt to reduce proteolysis, we have created a MB-1TrpHis gene library in which the two amino acids surrounding the peptide bond, N44 and L45, were randomized using degenerated oligonucleotides. The initial characterization of MB-1TrpHis N44E/L45V and MB-1TrpHis N44E/L45M, 2 variants of the library that were more resistant than the parent protein, was performed in order to investigate the nature of the mutants' resistance. Our results suggest that the mutants behaved like MB-1Trp regarding folding and thermal stability, and that proteolytic resistance is due to the elimination of the protease recognition site.

Cognitive empathy in inter-disciplinary research: the contrasting attitudes of plant breeders and molecular biologists towards rice.

I draw attention to the perceptions of and interactions between molecular biologists and scientists engaged in plant breeding in India, who have been attempting to employ molecular biology tools to understand and intervene to improve the rice crop. The present essay suggests that the concept of cognitive empathy is crucial for enabling basic scientists and applied scientists to begin to understand phenomena from the point of view of the other and from the point of view of society at large, and in arriving at solutions that are scientifically feasible and socially acceptable. Socialization into disciplinary cultures, organizational factors and individual anxieties seem to inhibit inter-disciplinary collaboration. The majority of rice breeders and a small group of molecular biologists emphasize the relative merits of marker-assisted selection (MAS) in the near term vis-à-vis the currently controversial transgenic approach for rice crop improvement in India.

Expression of a recombinant branched chain alpha-oxo acid dehydrogenase complex E2 (BCOADC-E2) in insect cells and its immunoreactivity to autoimmune sera

Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.

Protein engineering as a powerful tool for the chemical modification of enzymes

This article discusses the techniques of site-specific mutagenesis and protein engineering and their application in the study of enzyme active sites and the mechanism of enzyme action. Particular emphasis is given to beta-lactamase