High-level expression and characterization of single chain urokinase-type plasminogen activator (scu-PA) produced in recombinant Chinese hamster ovary (CHO) cells

2001 ◽  
Vol 6 (2) ◽  
pp. 117-127 ◽  
Author(s):  
Jung-Seob Kim ◽  
Mi-Kyung Min ◽  
Eui-Cheol Jo
Keyword(s):  
Plasminogen Activator ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Single Chain ◽  
High Level Expression ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
High Level

CHARACTERIZATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR MUTANTS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYS

1987 ◽  
Author(s):  
L Nelles ◽  
H R Linjnen ◽  
E Demarsin ◽  
D Collen ◽  
W E Holmes
Keyword(s):  
Plasminogen Activator ◽  
Specific Activity ◽  
Chinese Hamster ◽  
Expression Vectors ◽  
Wild Type ◽  
Single Chain ◽  
Plasma Clot ◽  
Serum Free ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

A cDNA encoding full length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced. The recombinant scu-PA (rscu-PA) cDNA as well as the cDNA of two mutants constructed by deoxyoligonucleotide directed mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158 ) or to Glu158 (rscu-PA-Glu158 ) were inserted into SV40 early promoter/enhancer based expression vectors, which were used to transfect Chinese Hamster Ovary (CHO) cells. The expression products were purified from serum-free conditioned media by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration.The amidolytic activity of the three rscu-PAs was low (< 500 IU/mg). The mutant rscu-PAs, in contrast to the rscu-PA and nscu-PA, could not be converted into an amidolytically active two-chain form (tcu-PA) by plasmin. The mutant scu-PAs had a very low specific activity (< 1,000 IU/mg) on fibrin plates, whereas wild type rscu-PA had a specific activity < 1000 IU/mg. The mutant scu-PAs did not cause lysis of a I-fibrin labeled plasma clot immersed in citrated human plasma. Serum-free medium from a control transfected CHO cell line showed no significant plasminogen activating activity.In a purified system, both rscu-PA-Gly and rscu-PA-Glu activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (K = 60-80 yM) but with a higher catalytic rate constant (k2 = B.01 s-1) as compared to the wild type rscu-PA (K =1.0 yM, k = 0.002 s-1).It is concluded thaz conversion of scu-PA to tcu-PA is prerequisite for the activation of plasminogen. However, Lys158 seems to be important for the stability of the Michaelis complex between scu-PA and plasminogen.

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