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* In this issue:

(1) Production of large heterotrimeric laminin glycoprotein in LEXSY
(2) Purification of proprotein convertase expressed in LEXSY
(3) Antibodies: Expression and purification of Fc fusions in LEXSY
(4) LEXSY products – all you need for cloning, expression, strain selection, and cultivation
(5) References

   
       
  
   
   

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    (1) Production of large heterotrimeric laminin glycoprotein in LEXSY
   


The protein expression platform LEXSY of Jena Bioscience was used to express human laminin-332 (α3β3γ2), a large heterotrimeric glycoprotein, an essential component of epithelial basal lamina that promotes cell adhesion and migration (Fig. 1).

The genes for the three individual chains were expressed from three LEXSY vectors making use of alternative selection markers available.



Figure 1: Model of heterotrimeric laminin-332 [1]


The recombinant LEXSY strain transfected with the three expression constructs efficiently formed the heterotrimer (145 + 135 + 150 kDa) and secreted it to the culture medium. The purified protein displayed full biological activity demonstrating proper folding and assembly including disulfide bonding.





Figure 2: Purification of laminin-332. 1 molecular size marker, 2 laminin from 293-F cells (2 forms), 3 laminin from LEXSY [2]



   
    (2) Purification of proprotein convertase expressed in LEXSY
   


Proprotein convertase 4 (PC4), a mammalian subtilase which plays a key role in fertilization was expressed in LEXSY, and soluble enzymatically active protein was purified from culture medium. Biochemical analysis demonstrated high specific activity (superior to PC4 from GH4C1 or Hi5 cells) and substrate specificity confirming its biological role and allowed inhibitor design for therapeutic and clinical applications [3].



   
    (3) Antibodies: Expression and purification of Fc fusions in LEXSY
   



Recombinant Fc fusions were expressed in LEXSY and one-step affinity purified from crude culture medium with Protein A sepharose. SDS PAGE analysis demonstrated that the proteins were secreted as dimers.






Figure 3: Purification of Fc fusion protein from LEXSY medium. 1 molecular size marker, 2-3 reducing conditions, 4 non-reducing conditions [4]



   
    (4) LEXSY products – all you need for cloning, expression, strain selection, and cultivation
   




Need further information? Please check out the LEXSY section at the Jena Bioscience website!

Or contact us by email at: expression@jenabioscience.com.


   
    (5) References
   


[1] www.unifr.ch/anatomy/elearningfree/francais/bindegewebe/funktion/popup_funktion/f-laminin.php (in french)

[2]    Phan et al. (2009) The production of recombinant human laminin-332 in a Leishmania tarentolae expression system. Protein Expression and Purification 68:79.

[3]    Basak et al. (2008) Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: Purification, biochemical study and inhibitor design. Protein Expression and Purification 60:117.

[4] Jena Bioscience GmbH (2009), not published.


   
   

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