Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.
BACKGROUND
VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant protein secretion from Pichia pastoris and if domain boundaries were applicable across different VAR2CSA alleles.


METHODS
VAR2CSA sequences were bioinformatically analysed to identify the predicted C11 and C12 cysteine residues at the C-termini of DBL domains and revised N- and C-termimal domain boundaries were predicted in VAR2CSA. Multiple construct boundaries were systematically evaluated for protein secretion in P. pastoris and secreted proteins were tested as immunogens.


RESULTS
From a total of 42 different VAR2CSA constructs, 15 proteins (36%) were secreted. Longer construct boundaries, including the predicted C11 and C12 cysteine residues, generally improved expression of poorly or non-secreted domains and permitted expression of all six VAR2CSA DBL domains. However, protein secretion was still highly empiric and affected by subtle differences in domain boundaries and allelic variation between VAR2CSA sequences. Eleven of the secreted proteins were used to immunize rabbits. Antibodies reacted with CSA-binding infected erythrocytes, indicating that P. pastoris recombinant proteins possessed native protein epitopes.


CONCLUSIONS
These findings strengthen emerging data for a revision of DBL domain boundaries in var-encoded proteins and may facilitate pregnancy malaria vaccine development.
Avril
Marion
M
Seattle Biomedical Research Institute, Seattle, Washington 98109-5219, USA. marion.avril@sbri.org
Hathaway
Marianne J
MJ
Cartwright
Megan M
MM
Gose
Severin O
SO
Narum
David L
DL
Smith
Joseph D
JD
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D003429:Cross Reactions / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D017780:Malaria Vaccines / Q000276:immunology*; D016778:Malaria, Falciparum / Q000276:immunology / Q000517:prevention & control; D010843:Pichia / Q000276:immunology / Q000378:metabolism; D010963:Plasmodium falciparum / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011110:Polymorphism, Genetic; D011247:Pregnancy; D015597:Pregnancy Complications, Parasitic / Q000097:blood / Q000276:immunology / Q000517:prevention & control; D015800:Protozoan Proteins; D011817:Rabbits; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C066001:Duffy antigen binding protein, Plasmodium; D000939:Epitopes; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C512601:VAR2CSA protein, Plasmodium falciparum