Merck Life Science, a division of Merck KGaA, Germany that offers a broad range of innovative, performance products, services and business relationships that enable our customers’ success in research, development and production of biotech therapies, pharmaceutical drug and vaccines. Through dedicated collaboration on new scientific and engineering insights, and as one of the top three R&D investors in the Life Science Tools industry, Merck Life Science serves as a strategic partner to customers and helps advance the promise of life science. Merck Life Science operates as Millipore Sigma in the U.S. and Canada.

Broad Area:

Development of recombinant vaccines against potential biowarfare agents

Bioprocess scale-up of recombinant biologicals

Work Place:Virology Division, Defence Research and Development Establishment, Gwalior, INDIA.

Dengue remains one of the most important arboviral disease. Control of dengue is a major goal. An effective vaccine against dengue that would protect nonimmune individuals from the disease has long been a dream. Immunity to dengue is complex, due to antibody mediated enhancement (ADE). Envelope protein of dengue virus is known to be highly immunogenic and has functional binding domain for membrane fusion and receptor mediated endocytosis.

I was leading a team involved in development of recombinant tetravalent sub-unit vaccine based on dengue envelope protein. We also worked on development of peptide based pan-flavivirus diagnostic system, development of detection system for early diagnosis of Kyasanur Forest disease. I worked towards development of bioprocess scale-up facility at DRDE, Gwalior. We did process scale-up (both upstream and downstream) for recombinant dengue multi-epitope protein for diagnostic use, recombinant protective antigen of Bacillus anthracis for prophylactic use.

Packing process scale chromatography column. Working in Scale-up facility of DRDO at Gwalior

Defence Research & Development Organisation (DRDO) works under Department of Defence Research and Development of Ministry of Defence. DRDO dedicatedly working towards enhancing self-reliance in Defence Systems and undertakes design & development leading to production of world class weapon systems and equipment in accordance with the expressed needs and the qualitative requirements laid down by the three services. DRDO is working in various areas of military technology which include aeronautics, armaments, combat vehicles, electronics, instrumentation engineering systems, missiles, materials, naval systems, advanced computing, simulation and life sciences. DRDO while striving to meet the Cutting edge weapons technology provides ample spinoff benefits to the society at large thereby contributing to the nation buliding.

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RESEARCH WORK DONE AT International Centre for Genetic Engineering & Biotechnology (ICGEB), NEW DELHI

Broad Area: Development of Recombinant Vaccine for Malaria

Work Place: Malaria Research Group, Intl Centre for Genetic Engineering & Biotechnol, New Delhi, INDIA.

View of International Centre for Genetic Engineering & Biotechnology, New Delhi

Preamble:

Malaria remains one of the most important vector borne human disease. Control of malaria caused by Plasmodium falciparum is a major goal. An effective vaccine against malaria that would protect nonimmune individuals from the disease has long been a dream. The life cycle of malaria parasite is complex, the several stages in humans are morphologically and antigenically distinct, and immunity is stage specific. The genome of P. falciparum probably contains about 5,000 genes, and only a subset of these is expressed at one stage. Erythrocyte invasion is an essential step in erythrocytic life cycle of Plasmodium falciparum. Erythrocyte invasion by P. falciparum requires the erythrocyte binding protein, EBA-175, which binds silalic acid residues present on glycophorin A of human erythrocytes. However, P. falciparum is known to possess alternate pathways for erythrocyte invasion. Sialic acid binding protein of P. falciparum has a putative signal sequence at the amino-terminus and a transmembrane segment followed by cytoplasmic domain at the carboxyl-end. The signal sequence is followed by a large extracellular domain that can be devided into six regions (region I to region VI). The extracellular domain contains two conserved cystein-rich regions (region II and VI). Region II of P. falciparum EBA-175 contains two copies of the amino-terminal cystein-rich region. Of the two cystein-rich domains (F1 and F2), only the second domain, F2 bound erythrocytes when expressed individually, indicating that it contains the binding sites. In our laboratory, we have observed that the functional recombinant erythrocyte binding domain (PfF2), expressed in E. coli offers protection from falciparum malaria as the antibody raised against rPfF2 blocked erythrocyte invasion by P. falciparum. Hence PfF2 has the potential to be used as vaccine candidate.

I was responsible for a project dealing with expression, refolding, large scale purification and scale-up of downstream processes towards industrial production and preclinical studies of recombinant Plasodium falciparum protein PfF2 (EBA-175, Vaccine candidate) AND associated with studies on receptor-ligand interaction of biomolecules involved in biology and pathogenesis of malaria.

Taking sample from fermenter. Working in Process Development Laboratory of ICGEB, New Delhi

The International Centre for Genetic Engineering and Biotechnology, as a part of the United Nations System, provides a scientific and educational environment of the highest standard and conducts innovative research in life sciences for the benefit of developing countries. It strengthens the research capability of its Members through training and funding programmes and advisory services and represents a comprehensive approach to promoting biotechnology internationally. The Centre is dedicated to advanced research and training in molecular biology and biotechnology and holds out the prospect of advancing knowledge and applying the latest techniques in the fields of: (1) biomedicine, (2) crop improvement, (3) environmental protection/remediation, (4) biopharmaceuticals and biopesticide production.

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Ph.D. RESEARCH WORK

Broad Area: Anaerobic Microbiology, Antimicrobial peptides, Bacteriocins

Title of the Project: Biochemical characterization and molecular genetics of bacteriocin produced by a selected rumen bacterial isolate.

Work Place: Molecular Biology Unit, National Dairy Research Institute, Karnal, INDIA.

Preamble:

Rumen represents a very complex ecosystem harboring a population of different microorganisms that interact and compete with each other for their survival. Efficiency of nutrient utilization in rumen is greatly dependent upon the balance of fermentation products, which is regulated by types and activities of rumen microorganisms. Manipulation of rumen function has always been a challenging task to improve the overall ruminant productivity. The recent strategy to enhance animal productivity is by manipulating rumen functionality by improvement of rumen bacteria through genetic engineering. Though a considerable research has already been directed towards genetic manipulation of rumen bacteria, all the experiments that have been carried out so far to transfer the engineegreen bacteria back into the competitive world of rumen have failed miserably.

In this context, bacteriocin production by rumen bacteria could be a very attractive and promising. They have distinct advantages over other options such as antibiotics, feed additives, etc. in view of their rumen origin, target specificity, susceptibility to proteolytic digestion, possibility of genetic transfer and relative ease of manipulation as well as safety considerations. Existence of extremely competitive interaction among rumen bacteria implies that they must be armed with the ability to produce bacteriocin and these bacteriocins may have significant impact on both the competitive fitness of individual microbial strains within the rumen and overall rumen microbial population structure. Rumen bacteriocins may find possible applications in minimizing methane emission from ruminants and controlling certain diseases like lactic acidosis and frothy bloat. All these features make bacteriocins from rumen bacteria, a potential candidate for detailed Biochemical and Molecular characterization before their possible in the rumen ecosystem. However, till today, only a handful of information concerning the occurrence of bacteriocins among rumen isolates or the sensitivity of rumen organisms to exogenous bacteriocins is available.

However, before exploring the potentials of these bacteriocins, we have to address few basic questions about the application of bacteriocins within the rumen environment. First, are endogenous rumen bacteriocins a significant factor in the rumen environment and second, can endogenous rumen bacteriocins or exogenous bacteriocins be useful for the manipulation of rumen populations in situ ?

Brief Summary of Research Findings:

To understand the molecular microbial dynamics of the rumen ecosystem, we studied bacteriocin (proteinaceous antibacterial compound) as a candidate to understand molecular behavior of amensalistic interaction. During the course of our study, we isolated a bacterium from buffalo rumen producing bacteriocin like substance as well as exhibiting strong hydrolytic activities. The isolate was later identified as Bacillus licheniformis 26L-10/3RA by 16S rRNA analysis. This is the first evidence of isolation of Bacillus licheniformis from buffalo rumen. Which indicates that other than obligatory anaerobes, facultative anaerobes like B. lichenmiformis can also survive in an anoxic environment and compete with other resident microflora for survival. The bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA was isolated and characterized to its molecular level. The antibacterial compound was identified as a highly hydrophobic peptide of 1.4 kDa molecular weight containing 12 amino acids. The antibacterial peptide was found to be a new molecule from the sequence homology study and was named as LICHENIN. The sequence of the peptide has been deposited to SwissProt data base under accession number P82907.

Lichenin exhibited broad spectrum antibacterial activity against a large number of diversified obligatory anaerobes including Streptococcus bovis SB3 and Ruminococcus albus B-199 only under anaerobic conditions. Surprisingly, lichenin was produced only under anaerobic condition was also active only under strict anaerobic conditions. This is the first report of anaerobiosis specific expression and production of any antibacterial substance which was active only at obligatory anaerobic condition. We found that the lichenin was extracellular and was produced at the late logarithmic and early stationary growth phase. Studies on optimization of cultural conditions for production of bacteriocin resulted maximal production in presence of inert solid substrates (thermocol beads). This observation provides further experimental evidence about contact regulation of production of secondary metablotes. Lichenin was characterized to be active at a wide range of pH 4.0-9.0, resistant to boiling for 15 min. oxygen labile and stable at room temperature.

The gene/genes encoding bacteriocin production was established to be located on chromosome. A genomic library was constructed using pUC18 vector and E. coli DH5 alpha host and the library was screened with biotin labeled degenerate probe.

National Dairy Research Institute, Indian Council of Agricultural Research, an autonomous Organisation under the Department of Agricultural Research and Education, Ministry of Agriculture, "Government of India". NDRI as the premier Research Institution undertakes research, teaching and extension activities towards dairy development in India. Being the National Institute, it conducts basic and applied research with the objective to enhance animal productivity and also to develop cost effective technologies. The institute has developed considerable expertise over last several decades in different areas of dairy Production ,Processing, Management and Human Resource development.

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MASTER DEGREE DISSERTATION WORK

Broad Area: Food Microbiology, Public health.

Title of the Project: Microbial load an microflora content of meat available at local market of Bhubaneswar.

Work Place: Department of Veterinary Bacteiology, Virology and Public Health, Orissa University of Agriculture and Technology, Bhubaneswar, INDIA.

Salient Findings:

Microbiological quality of randomly sampled market meat (mutton, chevon, beef, pork and chicken) with reference to total plate count, coliform count and yeast-mould count was determined and the meat samples ware found to contain high microbial load and were contaminated with high risk food pathogens. Studies on incidence of different toxigenic, non-toxigenic and spoilage microflora including fungi revealed that the spoilage microflora are predominant on raw meat. However, few samples yielded pathogenic serotypes of Salmonella species. Different bacteria and fungi was isolated from the meat samples and were identified and characterized.

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