Expressing Recombinant Coagulation Factors in Yeast and Insect Cells
J. Schwalbach, O. El-Maarri, U. Herbiniaux, P. Hanfland and J. Oldenburg
Introduction
Coagulation activity is controlled by a complex network of proteins. This cascade of proteins activation and deactivation is delicately controlled by protein interactions and by positive and/or negative feedbacks from end products. In this study we are expressing several coagulation factors (Factor II, VII, IX, X, AT3, factor V B domain and factor VIII domains) in e.coli, mammalian cells, yeast and insect cells based vector systems. The proteins will be used to select for specific aptamers against the surface of the protein molecules. As part of a novel microwave-array mass spectro- metry device these aptamers will be used for the qualitative and quantitative study of protein-protein interactions. RNA aptamers against factor IX have been shown as potential candidates for therapeutical use in anticoagulation (Rusconi et al. Nature 2002). Most interestingly, the antidot of the aptamer was represented by its anti- sense sequence.
Materials and Methods
We choose to use the Gateway system because of the easiness to shuttle the insert from one vector to another. The cDNAs were amplified from a human liver library using primers attached to attB1 and attB2 sequences (Fig. 1). After purification the PCR product was inserted by an enzymatic reaction (with BP enzyme mix from Invitrogen) into a pDONR Gateway vector (Invitrogen); the reaction produce more that 90% positive clones, after e.coli transformation, with the right insert orienta- tion. This would tremendously save time and efforts that would otherwise be need- ed when using normal cloning with restriction enzyme, especially when large cDNAs has to be cloned.
Once the cDNA were successfully inserted in the pDONR vector, the inserts were sequenced, following standard procedure (using ABI 310 machines), to verify the sequence integrity. The inserts were shuttled to pDEST 17 (e.coli), pBAD-DEST 49 (e.coli), pDEST 26 (mammalian), Baculovirus (SF9 insect cells) and pPICZa (yeast) and some to pGBKT7 (from Clonetech: converted to a gateway compatible vector) for protein interaction studies (Fig. 1).
The successful expression of the protein were than tested by both SDS page and western blot analysis. For western blot an antibody recognizing penta-His was used from QIAGEN (Fig. 2).
I. Scharrer/W. Schramm (Ed.)
34thHemophilia Symposium Hamburg 2003
” Springer Medizin Verlag Heidelberg 2005
Results and Discussion
We found that the Gateway platform is an easy system that would allow the shuttl- ing of a given insert to different expression vectors. This is particularly useful when many functional studies are to be performed.
The expression of human proteins in E.coli is not an easy and straightforward process as many eukaryotic proteins are not expressed by an e.coli vector. When using the pDEST 17 vector we were unable to get a signal on western blots, however by fusing the human protein with a protein that is expressed in both prokaryote and eukaryote (Thioredoxin in pBAD-DEST49) we were able to get good signals on Western blots, from the factor VIII domains, that correspond to the expected size of the fused protein. Moreover the pBAD-DEST49 expression can be tightly controlled by different concentrations of arabinose; we found that for most proteins a 0.02 to 0.002% arabinose concentration was optimum for the highest signal on western blots. Experiments with pDEST 26 in CHO cells are still underway as well as the yeast and the insect cell expression, however it is expected that the later approaches will give higher yields.
Expressing Recombinant Coagulation Factors in Yeast and Insect Cells 339
PCR product attB1-Gene-attB2
+ BP Enz + pDONR 201 attP1-ccdB-attP2
Entry clone attL1-Gene-attL2
Destination vectors attR1-ccdB-attR2
+LR Enz
E.coli expression:
pDEST 17 Yeast two Hybrid:
pGBKT7-G pGADT7-G
Expression clones attB1-Gene-attB2
Insect expression:
Baculovirus
Mammalian expression:
pDEST 26
Fig. 1. general scheme for vectors formations, the vectors used are mentioned.
340 J. Schwalbach et al.
Fig.2.SDS-PAGE gels (upper part) and western blots (lower parts) ofdifferent domains ofthe factor VIII protein expressed in pDAB-DEST 49 vec- tor that produce a hybrid protein fused with Thioredoxin at the N-terminal.
