U-Protein Express BV is specialized in the production of research grade and pre-clinical recombinant proteins and recombinant antibodies in mammalian expression systems. The basic principles of our technologies are explained below. If you need more information, please send us an email.

 

Recombinant protein production; r-PEx platform.

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Recombinant proteins are produced via the novel pipeline "r-PEx" to obtain large amount of research grade protein in a very short time span (generally less than 8 weeks). The pipeline from gene to purified protein comprises the following steps:r-PEx technology

 

1. Generation of coding cDNA via synthetic gene design and codon optimization or PCR.

2. Ligation of the coding sequence in several expression vectors.

3. Small scale transient protein production to suspension growing HEK293 cells. Three to 6 days post transfection recombinant protein production is assayed and the most optimal expression vector is selected.

4. Large scale transient protein production in suspension growing HEK293 cells.

5. Purification and characterization of the recombinant protein.

The advantage of the r-PEx transient expression platform is that the recombinant protein production can easily be switched to lower expression platforms like the yeast Pichia pastoris or the bacterium Escherichia coli, which both are available at U-protein Express BV.

 

Expression vector design

Some characteristics of the pUPE expression vectors are listed below. Due to the modular architecture the expression vectors can easily be adapted to meet specific requirements.

  • Internal or secreted protein production
  • Standard restriction sites (5'-BamHI and 3'-NotI)
  • Large number of purification tags
    • Hexahistidine, Strep, Fc, FLAG
    • With or without TEV-protease cleavage site

 

 

Project options

The r-PEx platform has a modular architecture. Therefore the client has at least the following transfection and delivery options:

Options for DNA preparation and transfection
Option 1 Transfection of client DNA.
The client provides sufficient amount of transfection grade plasmid DNA containing protein cDNA in client expression vector.
Option 2

Transfection of client DNA.
The client ligates protein cDNA in pUPE expression vector and provides sufficient amount of transfection grade plasmid DNA (license required).

Option 3 Client delivers plasmid DNA. U-ProteinExpress prepares transfection grade plasmid DNA.
Option 4 U-ProteinExpress ligates cDNA in 1 pUPE expression vector and transfection grade plasmid preparation and transfection.
Option 5 U-ProteinExpress ligates cDNA in 6 pUPE expression vectors, selection of optimal expression vector, transfection grade plasmid preparation and transfection.
Options for delivery
Option 1 Delivery of cleared expression medium.
Option 2 Delivery of concentrated expression medium with buffer exchange.
Option 3 Delivery of purified protein with 1 step purification.
Option 4 Delivery of purified protein with >1 step purification.

 

 

  

Recombinant antibody production r-Ab platform.

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At U-Protein Express BV fully post-translational modified mammalian recombinant antibodies (rAb’s) are produced via its r-PEx transient expression platform (patent applications pending). The r-PEx expression platform is based on Human Embryonic Kidney cells (HEK293E) and yields 40 - 150 mg/L amount of recombinant antibody. Our maximal capacity is 60 L, resulting in 2.5-9 gram of purified rAb per batch per week. Transient production of ADCC-enhanced recombinant antibodies is available using CHO-Potelligent cells from BioWa. Expression levels of recombinant antibodies in CHO-Potelligent is about 0.5 - 1.0 mg/L. Using dedicated protocols the endotoxin level in a purified rAb batch is very low, less than 0.5 EU/mg. Typical turnaround times from hybridoma to purified rAb is 10 weeks.

rAb production technology

 

 

The recombinant antibody production suite comprises the following steps:

 

  1. Isolation of mRNA coding for the antibody heavy and light chains and conversion into cDNA
  2. Sequencing of the antibody variable domains and ordering codon-optimized genes
  3. Ligation of variable domains into full-length antibody expression vectors of any class and of any organism
  4. Transient production of rAb in HEK293E or CHO-Potelligent cells via the r-PEx technology
  5. Purification of the rAb via affinity chromatography and gel filtration chromatography
  6. Assaying the function of the purified rAb

 

 

 

 

 

 

 

Production of recombinant antibodies has numerous advantages compared to traditional antibody production. For example, 

 

 

  1. Reproducible and high production yield
  2. (Sub-)class swithces are possible
  3. Specific effector functions can be changed by the introduction of mutations in the constant or variable domains
  4. The rAb can be produced in fucosylation deficient mammalian expression hosts, thereby increasing ADCC
  5. There is no need to store hybridoma's

 

Project options

The r-Ab platform has a modular architecture. Therefore the client has the following transfection and delivery options:

Options for DNA preparation
Option 1

Client delivers transfection grade plasmid DNA.

Option 2 Client ligates variable domains in pUPE antibody expression vectors and provides sufficient amount of transfection grade plasmid DNA (license required).
Option 3 Client delivers plasmid DNA. U-ProteinExpress prepares transfection grade plasmid DNA.
Option 4 Client provides U-ProteinExpress with sequence of variable domains.
U-ProteinExpress orders codon-optimized variable domains, ligation into antibody expression vectors.
Option 5

Client provides U-ProteinExpress with hybridoma.
U-ProteinExpress determines antibody sequences, orders codon-optimized variable domains, ligation into antibody expression vectors.

Options for transfection and delivery
Option 1 Transfection up to 50 liter and delivery of cleared expression medium.
Option 2 Transfection up to 50 liter and delivery of concentrated expression medium with buffer exchange.
Option 3 Transfection up to 50 liter and delivery of proteinA purified antibody.
Option 4 Transfection up to 50 liter and delivery of proteinA and gelfiltration purified antibody.