Natarajan Velmurugan

Recombinant antibodies: engineering and production in yeast and bacterial hosts.

After the appearance of the first FDA‐approved antibody 25 years ago, antibodies have become major therapeutic agents in the treatment of many human diseases, including cancer and infectious diseases, and the use of antibodies as therapeutic/diagnostic agents is expected to increase in the future. So far, a variety of strategies have been devised for engineering of these fascinating molecules to develop superior properties and functions. Recent progress in systems biology has provided more information about the structures and cellular networks of antibodies, and, in addition, recent development of biotechnology tools, particularly in regard to high‐throughput screening, has made it possible to perform more intensive engineering on these substances. Based on a sound understanding and new technologies, antibodies are now being developed as more powerful drugs. In this review, we highlight the recent, significant progress that has been made in antibody engineering, with a particular focus on Fc engineering and glycoengineering for improved functions, and cellular engineering for enhanced production of antibodies in yeast and bacterial hosts.

Evaluation of intracellular lipid bodies in Chlamydomonas reinhardtii strains by flow cytometry.

A comparative study of Chlamydomonas reinhardtii wild type CC124 and a cell wall-less mutant sta6-1 is described using FACS in conjunction with two different lipophilic fluorescent dyes, Nile Red and BODIPY 505/515. The results indicate that BODIPY 505/515 is more effective for the vital staining of intracellular lipid bodies and single cell sorting than Nile Red. While BODIPY 505/515 stained cells continued to grow after single cell sorting using FACS, Nile Red stained cells failed to recover from sorting. In addition, a comprehensive study was performed to establish a quantitative baseline for future studies for either lipid accumulation and/or microalgal growth by measuring various parameters such as cell count, size, fatty acid contents/composition, and optical/confocal images of the wild type and mutant.

Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in Chlamydomonas reinhardtii.

BackgroundThe concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions.ResultsThe successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution.ConclusionsAdaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.

Study of cellular development and intracellular lipid bodies accumulation in the thraustochytrid Aurantiochytrium sp. KRS101

Transmission electron, confocal microscopy and FACS in conjunction with two different lipophilic fluorescent dyes, BODIPY 505/515 and Nile Red were used to describe the cellular development and lipid bodies formation in Aurantiochytrium sp. KRS101. TEM results revealed that multi-cellular spores were appeared in sporangium during early-exponential phase, and spores were matured in mid-exponential phase followed by release of spores from sporangium in late-exponential phase. TEM and FACS analyses proved that lipid bodies appeared, developed and degenerated in mid-exponential, early- and late-stationary phases, respectively. The staining results in FACS indicate that BODIPY 505/515 is more effective for the vital staining of intracellular lipid bodies than Nile Red. FACS based single cell sorting also showed healthy growth for BODIPY 505/515 stained cells than Nile Red stained cells. In addition, a quantitative baseline was established either for cell growth and/or lipid accumulation based on cell count, fatty acid contents/composition, and sectional/confocal images of KRS101.

Effect of low shear modeled microgravity on phenotypic and central chitin metabolism in the filamentous fungi Aspergillus niger and Penicillium chrysogenum

Phenotypic and genotypic changes in Aspergillus niger and Penicillium chrysogenum, spore forming filamentous fungi, with respect to central chitin metabolism were studied under low shear modeled microgravity, normal gravity and static conditions. Low shear modeled microgravity (LSMMG) response showed a similar spore germination rate with normal gravity and static conditions. Interestingly, high ratio of multiple germ tube formation of A. niger in LSMMG condition was observed. Confocal laser scanning microscopy images of calcofluor flurophore stained A. niger and P. chrysogenum showed no significant variations between different conditions tested. Transmission electron microscopy images revealed number of mitochondria increased in P. chrysogenum in low shear modeled microgravity condition but no stress related-woronin bodies in fungal hyphae were observed. To gain additional insight into the cell wall integrity under different conditions, transcription level of a key gene involved in cell wall integrity gfaA, encoding the glutamine: fructose-6-phosphate amidotransferase enzyme, was evaluated using qRT-PCR. The transcription level showed no variation among different conditions. Overall, the results collectively indicate that the LSMMG has shown no significant stress on spore germination, mycelial growth, cell wall integrity of potentially pathogenic fungi, A. niger and P. chrysogenum.

High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli

For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e–GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e–GST with highest solubility were achieved with E. coli BL21(DE3) at 25xa0°C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11xa0g of VP1e–GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.

Novel strategy for production of aggregation-prone proteins and lytic enzymes in Escherichia coli based on an anchored periplasmic expression system

For over 2 decades, Escherichia coli has been successfully used for the production of various recombinant proteins. However, several technical limitations have influenced the extent of recombinant protein expression in the E.xa0coli host because of (i) heterologous protein accumulation often observed in inactive inclusion bodies either in the cytoplasm or periplasm, or (ii) lytic activity of recombinant proteins, which causes cell lysis, that hinder high production yield. We developed a novel strategy for the efficient production of aggregation-prone proteins and lytic enzymes in the E.xa0coli host. For this purpose, we used an anchored periplasmic expression (APEx) system, in which target proteins are produced in the periplasm and tethered on the inner membrane. Protein aggregation and lytic activity can be prevented through anchoring of individual proteins to the inner membrane. Two model proteins (aggregation-prone human leptin and lytic Pseudomonas fluorescens SIK W1 lipase) were examined, and both proteins were successfully produced and anchored to the inner membrane under optimized culture conditions. Upon expression, the inner membrane-anchored proteins were subjected to simple purification procedures; the proteins were confirmed to be of high purity and bioactivity.

Enhanced production of human FcγRIIa receptor by high cell density cultivation of Escherichia coli

Human Fc receptors (FcγR) are membrane glycoproteins that are expressed on all immunologically active cells and have a well-defined role in regulating innate and adaptive immune responses by binding to the immunoglobulin G (IgG) antibody. Among the several classes of Fc receptors, FcγRIIa is the most widely expressed, and it serves as an important reagent in antibody engineering. Here, we report on high cell density cultivations (HCDC) of Escherichia coli for preparative scale production of FcγRIIa in a 6.6L bioreactor. Briefly, a pH-stat feeding strategy was employed, and two different cell densities (OD(600) of 46 and 100) were examined for the induction of FcγRIIa gene expression. When cells were induced at a high cell density (OD(600) of 100), the cell density increased to an OD(600) of 234 within 9h after induction, and a 2-fold higher production yield was obtained compared with that of induction at low cell density (OD(600) of 46). After simple purification steps including denaturation and refolding, 87.7 mg of soluble FcγRIIa that was more than 95% pure was obtained from a 20-mL culture with high recovery yield (≈54%). The biological activity of purified FcγRIIa was also confirmed by evaluating its interaction with all subclasses of IgG antibodies using an ELISA bioassay.