Expression of a recombinant anticoagulant C-type lectin-like protein ACFI in Pichia pastoris: heterodimerization of two subunits is required for its function.
ACFI is an anticoagulant C-type lectin-like protein (CLP) isolated from Agkistrodon acutus venom. To investigate the function of ACFI and its subunits, the cDNAs of two subunits were transformed and expressed in Pichia pastoris separately or together by a novel strategy using two vectors with different selectable markers. The results showed that recombinant homodimers were secreted when the subunits were expressed alone, while heterodimers (rACFI) were secreted when two subunits were co-expressed. The secreted proteins were purified from culture supernatants in one step by metal-chelating affinity chromatography with the yields of 1-4 mg/L. PAGE and ELISA showed that rACFI competed the binding of native ACFI for human factor X and IX with affinities of 1.6 and 30 nM, respectively. In addition, rACFI prolonged the activated partial thromboplastin time (APTT) in a concentration dependent manner as same as native ACFI. However, recombinant alpha or beta homodimers completely lost these activities, indicating the heterodimerization of two subunits is required for its function. It also suggests that P. pastoris is a promising system for structure-function studies of snake CLPs.
Hu
Siyi
S
Life Science School, University of Science & Technology of China, Hefei 230027, Anhui, People's Republic of China.
Li
Weifang
W
Chen
Lan
L
Liu
Jing
J
D017836:Agkistrodon / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D015870:Gene Expression; D037181:Lectins, C-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D037181:Lectins, C-Type; D021122:Protein Subunits