Joseph Shiloach, Ph.D.
Biotechnology Core, Cores and Support Services
Building 14A, Room 173
14 Service Rd West
Bethesda, MD 20814
+1 301 496 9719
The research purpose is to develop efficient methodologies for production of various biological materials, such as proteins and viruses, from different biological sources, especially microorganisms and mammalian cells. These biological products are being used for basic research, structural studies and clinical applications directed toward the development of effective new drugs and vaccines.
The laboratory’s main interest is production, recovery and purification of biologicals from both prokaryotes and eukaryotes of native and recombinant sources. The work involves all aspects of this process including research, development, and production. In addition to research labs, the laboratory includes pilot production facility, well equipped with bioreactors of different sizes and variety of recovery and purification equipment. The bioreactors are used for propagation of biological systems such as mammalian cells, insect cells, bacteria, yeast, and fungi. The biological products made support clinical and structural studies.
We conduct research and process development on growth optimization, production, scale-up, and product-recovery processes. We concentrate on production and purification of proteins with emphasis on scaling up. The aim of our research studies is to achieve better understanding of the growth behavior and metabolism of both E. coli and several mammalian cells lines. The objective is to overcome specific difficulties in the growth and production process by modifying the properties of the producers. This work involves gene transcription and expression analysis as well as cell transfection and mutation. Our recent achievements are summarized below:
- We identified genes involved in mammalian cell adhesion, which allows us to modify the way the cells grow. The gene Siat7e was overexpressed in the adherent MDCK cells and, as a result, the cells were able to grow in suspension. These cells were then utilized for the preparation of influenza vaccine replacing the current influenza vaccine production, which is based on using fertilized eggs.
- We identified microRNAs that are involved in controlling apoptosis onset in mammalian cells, allowing us to extend the growth phase of the cells and potentially enhance the production of biological compounds. Inhibition of the microRNA 466h increased the expression levels of anti-apoptotic genes and increased the cell viability.
- We improved glucose utilization by E. coli through the over-expression of small RNA. This was responsible for glucose transport, allowing the bacteria to grow to high density. Over expression of the small RNA sgrS in E. coliK strain reduced the level of the glucose transporter PTSG and allowed the bacteria to increase the glucose utilization yield, reduce acetate production, and to increase the bacteria concentration and recombinant protein production.
- By expanding our work related to noncoding RNAs we conducted High-Throughput screening of approximately 800 microRNA molecules. By careful analysis we identified 2 microRNAs that improved recombinant protein expression form HEK293 cell. This approach was initiated when large quantities of neurotensin receptor, hard to express proteins, were needed for structural studies.
- The high throughput screening approach of microRNA was expanded to screen more than 23000 genes by utilization human siRNAs library. After extensive analysis 10 genes whose inactivation was associated with improved recombinant protein expression were selected for further evaluation. The top candidate “Ornithine decarboxylase antizyme” (OAZ1) was knocked out using crisper technology to create an improved producing cell line.
- We developed a continuous production process of retroviral vector needed for adoptive T-cell Therapy. The process is based on growing the producing cells (PG13) on microcarriers in stirred tank bioreactor equipped with alternative tangential flow perfusion system. No detrimental effects on the specific viral vector titer or on the efficacy of the vector transducing the T- cells of several patients. Viral vector titer increased throughout the 11 days of the perfusion period. An indication that this method can be an efficient way to produce large quantities of active vector suitable for clinical use.
- Improved expression of protective antigen (PA) from Bacillus anthracis ames strain BH500. We developed newer approach to conduct extensive RNA seq analysis of samples from expressing and non-expressing Bacillus strain. As a result, we were able to identify key genes (not identified before) whose modified expression may improve the PA production.
Applying our Research
Improved methodologies for production and purification of biological compounds intended for clinical research will provide researchers with the materials needed for the evaluation and testing of potential new drugs and vaccines against various diseases.
- Adjunct Professor, Johns Hopkins University, Department of Chemical and Biochemical Engineering
- Associate Professor, Georgetown University - Biochemistry and Molecular & Cellular Biology
- Research Associate, Tufts University, New England Enzyme Center, 1975-1978
- Ph.D., Hebrew University, Israel, 1975
Inwood S, Xu H, Black MA, Betenbaugh MJ, Feldman S, Shiloach J. Continuous production process of retroviral vector for adoptive T- cell therapy. Biochem Eng J. 2018;132:145-151.
Inwood S, Buehler E, Betenbaugh M, Lal M, Shiloach J. Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen. Biotechnol J. 2018;13(2).
Xiao S, Chen YC, Buehler E, Mandal S, Mandal A, Betenbaugh M, Park MH, Martin S, Shiloach J. Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells. Biotechnol Bioeng. 2016;113(11):2403-15.
Liu WC, Zhou F, Xia D, Shiloach J. Expression of multidrug transporter P-glycoprotein in Pichia pastoris affects the host's methanol metabolism. Microb Biotechnol. 2019.
Sharma AK, Leppla SH, Pomerantsev AP, Shiloach J. Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500. Sci Rep. 2018;8(1):16108.
Related Scientific Focus Areas
Genetics and Genomics
Microbiology and Infectious Diseases
Molecular Biology and Biochemistry
This page was last updated on October 15th, 2020