Transplantation Biology Research Center

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Recombinant Protein Expression and Purification Core

The Recombinant Protein Expression and Purification (RPrEP) Core in the Transplantation Biology Research Center at Massachusetts General Hospital provides cost-effective resources for expression and purification of recombinant proteins necessary for preclinical and basic mechanistic studies by immunologists and cancer biologists.

The Recombinant Protein Expression and Purification (RPrEP) Core in the Transplantation Biology Research Center (TBRC) at Massachusetts General Hospital was established in 2009 to provide a cost-effective resource to the research community. RPrEP provides expression and purification of recombinant proteins necessary for preclinical studies as well as for basic mechanistic studies by immunologists and cancer biologists.

Recombinant proteins can be manufactured by this Core for a variety of applications according to an investigator’s needs. In addition to large-scale production of recombinant proteins in yeast Pichia pastoris, we provide a cost effective, small-scale production service using E. coli or yeast Pichia Pastoris expression system. This system could be used in small animal studies as immunogens, ELISAs or high throughput microarray platforms.

The main interest and expertise of the RPrEP Core is using a unique diphtheria toxin-resistant yeast Pichia pastoris expression system as a platform to develop diphtheria toxin-based recombinant immunotoxins/fusion toxins for specific depletion of targeted cell populations in vivo.

The Core has developed:

  • Anti-porcine CD3 immunotoxin for depleting porcine CD3 T cells in vivo 
  • Porcine CTLA-4 fusion toxin for depleting porcine CD80+ or CD86+ antigen presenting cells in vivo
  • IL-2 fusion toxins (human, porcine and murine versions) for depleting IL-2R+ cells including CD25+ Tregs in vivo

IL-2 fusion toxins can be used for studying the role of Tregs in autoimmune diseases, transplantation tolerance and cancer immunotherapy, as well as for directly depleting CD25+ tumor cells such as CD25+ cutaneous T cell lymphoma.  All of these novel fusion toxins are available through the RPrEP Core for research purposes, and additional fusion toxins can be developed upon request.
Research Projects

Currently, we have two ongoing projects involving the development of novel recombinant immunotoxins. The first is to develop an anti-human CCR4 immunotoxin. It was reported that the majority of Tregs express the chemokine receptor CCR4. Tumor cells express chemokine CCL17 (TARC) and CCL22 (MDC), which attract CCR4+ Tregs to the tumor local environment to evade the host immune system attack. We are developing a diphtheria toxin-based anti-human CCR4 immunotoxin to:

  • Facilitate studies of cancer treatment via specifically depleting CCR4+ Tregs
  • Directly deplete human CCR4+ cancer cells such as CCR4+ cutaneous T-cell lymphoma
  • Treat allergic diseases such as asthma via depleting type 2 helper T cells (CCR4+ Th2 cells) and invariant natural killer T cells (CCR4+ iNKT cells)

Our second ongoing project is the development of porcine CD40L fusion toxin by targeting porcine CD40+ antigen presenting cells and porcine CD40+ tumor cells. 

Core Leader
Zhirui Wang, DVM, PhD
Core Leader/Technical Director, RPrEP Core, TBRC
Assistant Immunologist, Massachusetts General Hospital
Assistant Professor of Surgery, Harvard Medical School
(617) 643-1957
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Email

Zhirui Wang, DVM, PhD, completed his doctorate in molecular biology from Justus-Liebig-University of Giessen in Germany in 1995. Dr. Wang completed his Doctor of Veterinary Medicine training in 1982, and his Master of Science in Veterinary Preventive Medicine in 1988 from Shanxi Agricultural University in China. He gained expertise in protein engineering and targeted drug delivery systems through post-doctoral training in the laboratory of David Neville, MD, at the National Institutes of Health, before joining the Mass General Transplantation Biology Research Center in 2007.

Currently, Dr. Wang holds appointments as assistant professor of surgery at Harvard Medical School and assistant immunologist at Mass General. Under Dr. Wang’s supervision, the Recombinant Protein Expression and Purification (RPrEP) Core has successfully completed more than 70 recombinant protein expression and purification projects for Mass General and DF/HCC investigators.

Scientific Director
David H. Sachs, MD

Scientific Co-Director
Christene A. Huang, PhD

Research Fellow
Hongyuan Chen, PhD

Technical Personnel
Zhaohui Wang, DVM
Huiping Zhang

Accounts Manager
Pat Kiszkiss

Below are lists of available services and equipment provided by the Recombinant Protein Expression and Purification Core, as well as a link to our fee schedule. Access to the Core is available to all members of the research community. Priority will be given to Massachusetts General Hospital and Dana Farber/Harvard Cancer Center users. 

Services

Bio-Rad Gene-Pulser X-Cell TBRC

Bio-Rad Gene-Pulser X-Cell for yeast transformation.

  • Recombinant protein expression and purification in yeast Pichia Pastoris
    • Codon optimization for yeast Pichia Pastoris expression 
    • Synthesis of entire codon-optimized gene of interest 
    • Cloning/subcloning of the codon-optimized gene of interest into a yeast expression vector 
    • Transformation of yeast and selection of transformants 
    • Verification of expression of the gene of interest by SDS gel and Western blot 
    • Small-scale expression from one to three liters 
    • Development of efficient purification strategy 
    • Scale-up expression using BioFlo 510 fermentor
  • Cost effective small-scale expression and purification in E. coli system 
  • Large-scale purification of recombinant protein
  • Large-scale production of immunotoxins for cell depletion studies
  • Large-scale production of recombinant cytokines (e.g. IL-2, IL-3) not available or prohibitively expensive from commercial sources
  • Yeast display for protein-protein/antibody interaction study
  • BioFlo 510 Fermentor training and usage

Equipment

Equipmentmanufacturer
BioFlo 510 Fermentor:19.5L New Brunswick Scientific
HPLC System Shimadzu
Refrigerated Orbital Shaking Incubators (3) New Brunswick Scientific
Bio-Rad GenePulser X-Cell BioRad
Spectrophotometer BioRad
Large scale purification columns (Index 100/500 column) GE Healthcare
GeneAmp PCR System 9700 Applied Biosystem
Refrigerated Centrifuge 5417R Eppendorf
Refrigerated Centrifuge Legend RT Sorvall
The Endosafe-PTS Portable Test System Charles River Laboratories
Refrigerated Centrifuge 5417R Eppendorf

Fee Schedule

For current information on pricing, please visit our page on the Partners Research Core Facilities website.

Yeast display vector: pYD1, pYD5, and yeast display strain: yeast Saccharomyces cerevisiae EBY100 are available in this core (redistribution of pYD1, pYD5 and EBY100 permitted by inventors) for the research community.

Location

The RPrEP Core facility is located at:

Massachusetts General Hospital
Charlestown Navy Yard
Building 149, 13th Street
Charlestown, MA 02129-2020