Changing the N-terminal sequence protects recombinant Plasmodium falciparum circumsporozoite protein from degradation in Pichia pastoris.
Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP)( n ) repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.
Zhang
Qingfeng
Q
Department of Pathogen Biology and State Key Laboratory of Medical Immunology, Second Military Medical University, 800 Xiang Yin Road, Shanghai, 200433, China.
Ding
Feng
F
Xue
Xiangyang
X
Xu
Xindong
X
Pan
Weiqing
W
D000818:Animals; D004655:Emulsions; D008353:Mannitol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009829:Oleic Acids / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004655:Emulsions; D009829:Oleic Acids; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041595:circumsporozoite protein, Protozoan; C001681:mannide monooleate; D008353:Mannitol