Recombinant Protein Expression and Purification Core
Scientific Director: Dr. David Sachs
Location: Building CNY 149-6117C 13th St. Charlestown, MA 02129


 
 
Core Summary:

The Recombinant Protein Expression and Purification (RPrEP) Core was established in 2009 to provide a cost-effective resource to the MGH and DF/ HCC community for expression and purification of recombinant proteins necessary for pre-clinical 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 MGH-DF/ HCC investigator needs. In addition to large-scale production of recombinant proteins in yeast Pichia pastoris, a cost effective small-scale production service will be provided using E.coli or yeast Pichia pastoris expression system (e.g. for small animal studies, use as immunogens, ELISAs, or high throughput microarray platforms).

Personnel/Contact Information:

Personnel:

  • Scientific Director: Dr. David H. Sachs
  • Scientific Co-director: Dr. Christene A. Huang
  • Technical Director: Dr. Zhirui Wang
  • Accounts Manager: Pat Kiszkiss

Contact Information:

Technical Director: Dr. Zhirui Wang
Phone: (617) 643-1957
Email: zhirui.wang@tbrc.mgh.harvard.edu

Facilities & Equipment:

Location of Core:

The facility is located at CNY 149-6117C 13th St. Charlestown, MA 02129.

Equipment:

Equipment
Manufacturer
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

 

Services:

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

*Access to the Core will be available to all members of the research community. Priority will be given to MGH and DF/HCC users.  

Fee Schedule/Rates:

 

Rates for MGH:
(NonMGH academic investigators will be charged for 44% indirect cost, Industry - please inquire about pricing)

Cat. #

Protein Name

Host

Price

RP1

NHP anti-CD3 immunotoxin

P. pastoris

$1,000/mg

RP2

Porcine anti-CD3 immunotoxin

P. pastoris

$625/mg

RP3

Porcine IL3-Gly

P. pastoris

$500/mg

RP4

Human Annexin A2-Gly

P. pastoris

$250/mg

RP5

NHP IL3-Gly

P. pastoris

$250/mg

RP6

NHP IL3-NonGly

P. pastoris

$250/mg

RP7

Porcine IL3-NonGly

P. pastoris

$278/mg

RP12

Human IL2 *

P. pastoris

$500/mg

RP13

Mouse IL2 *

P. pastoris

$500/mg

RP15

pYD5 (yeast display plasmid vector)

 

$250/vial

RP16

Porcine CTLA4-Gly

P. pastoris

$625/mg

RP17

Human Annexin-A2-NonGly

P. pastoris

$278/mg

RP18

Porcine CTLA4-NonGly

P. pastoris

$625/mg

RP22

Porcine IL2-Gly *

P. pastoris

$500/mg

RP23

Porcine IL2-NonGly *

P. pastoris

$500/mg

RP24

pYD1 (yeast display plasmid vector)

 

$250 /vial

RP25

yeast Saccharomyces cerevisiae EBY100

 

$250 /vial

RP27

DT390-pCTLA4-NonGly

P. pastoris

$625/mg

RP28

DT390-bi-pCTLA4-NonGly

P. pastoris

$625/mg

RP34

DT390-bi-pIL2-NonGly

P. pastoris

$625/mg

RP38

DT390

P. pastoris

$625/mg

RP39

DT390-bi-hIL-2

P. pastoris

$715/mg

RP42

DT390-hIL-2

P.pastoris

$715/mg

RP43

DT390-pIL-2-NonGly

P. pastoris

$625/mg

RP46

Soluble porcine CTLA4-L134M

P. pastoris

$715/mg

RP48

Soluble mouse CTLA4

P. pastoris

$715/mg

RP49

Porcine IL-15

E. coli

$1000/mg

RP50

Soluble human CTLA4

P. pastoris

$715/mg

RP55

Porcine IL-10

P. pastoris

$278/mg

RP56

DT390-mIL2

P. pastoris

$715/mg

RP57

Human IL-15

E. coli

$1000/mg

RP58

Murine RSPO2

P. pastoris

$715/mg

 

Recombinant Protein Development

P. Pastoris

 

$5000 (see description)

 

 

 

* Price listed is for orders of 1mg or greater. For orders less than 1mg, price is $180/100µg.

Recombinant Protein Development Service Description

Amount

Development of recombinant yeast expression protocol and purification strategy including:

$5,000

Codon optimization for expression in pichia pastoris system

Synthesis of entire codon optimized gene

Sequence confirmation

Cloning the gene into a yeast expression vector

Transformation of yeast and selection of transformants

Development of the expression protocol using shaker flasks

Verification of the expression by SDS gel and Western blot analysis

NOTE: If the expression level is too low or the protein cannot be solubilized, project may be terminated at this stage and investigator will be charged $2500 for service performed

Purified material from 2 L expression run using shaker flasks for functional testing

(No Charge)

Total

$5,000

The amount of material supplied depends on expression efficiency and yield after purification

Cost of additional protein to be determined based on expression level and yield

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 worldwide research community.

Potent IL-2 fusion toxins (murine, human and porcine) are available in this core for depleting IL-2 receptor positive cells including CD25+ Treg in vivo.

Core Publications:

[1] Hermanrud, C.E., Lucas, C.L., Sykes, M., Huang, C.A., Wang, Z. (2011) Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris. Protein Expr Purif. 76:115–120.

[2] Wang, Z., Duran-Struuck, R., Crepeau, R., Matar, A., Hanekamp, I., Srinivasan, S., Neville, D.M., Sachs D.H., Huang CA. (2011) Development of a Diphtheria Toxin Based Antiporcine CD3 Recombinant Immunotoxin. Bioconjug Chem. 22:2014-2020.

[3] Hermanrud, C. E., Pathiraja, V., Matar, A., Duran-Struuck, R., Crepeau, R.L., Srinivasan, S., Sachs, D. H., Huang, C. A., Wang, Z. (2012) Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris. Protein Expr Purif. 82:70-74.

[4] Peraino, J.,  Zhang, H., Hermanrud, C. E.,  Li, G., Sachs, D. H.,  M.D.; Christene A Huang, C. E., Wang, Z. (2012) Expression and purification of soluble porcine CTLA‑4 in yeast Pichia pastoris. Protein Expr Purif. 82:270-278.

[5] Peraino, J. S., Hermanrud, C. E., Springett, L., Zhang, H., Li, G., Srinivasan, S., Gusha, A., Sachs, D. H., Huang, C. A., Wang, Z. (2012) Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15. Cell Immunol. 276:162-167.

[6] Peraino, J. S., Schenk, M.,  Zhang, H., Li, G., Hermanrud, C. E., Neville Jr., D. M., Sachs, D. H., Huang, C. A., Duran-Struuck, R., Wang, Z. (2013) A Truncated Diphtheria Toxin Based Recombinant Porcine CTLA-4 Fusion Toxin. J Immunol Methods. 391:103-11.

[7] Peraino, J. S., Zhang, H., Li, G., Huang, C. A., Wang, Z. (2013) Molecular Basis of Cross-Species Reactivities of human versus Porcine CTLA-4. Hum Immunol.72:842-848.

[8] Peraino, J. S., Schenk, M., Li, G., Zhang, H., Farkash, E. A., Sachs, D. H., Huang, C. A., Duran-Struuck, R., Wang, Z. (2013) Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells. J Immunol Methods. doi:pii: S0022-1759(13)00246-9. 10.1016/j.jim.2013.09.006.

Getting Started:

To access the core, please contact Dr. Zhirui Wang at (617) 643-1957 or by email at
zhirui.wang@tbrc.mgh.harvard.edu.


 
 
     
 

 

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Elaine Zive
Associate Director of Research Core Facilities
EZIVE@PARTNERS.ORG
617-954-9851