Affinity
precipitation of human serum albumin using a thermo-response polymer with an
L-thyroxin ligand
Zhaoyang
Ding and Xuejun
Cao
BMC
Biotechnology 2013, 13:109 doi:10.1186/1472-6750-13-109
Published:
17 December 2013
Background
Affinity
precipitation has been reported as a potential technology for the purification
of proteins at the early stage of downstream processing. The technology could
be achieved using reversible soluble-insoluble polymers coupled with an affinity
ligand to purify proteins from large volumes of dilute solution material such
as fermentation broths or plasma. In this study, a thermo-response polymer was
synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl
acrylate as monomers. The molecular weight of the polymer measured by the
viscosity method was 3.06 x 104 Da and the lower critical solution temperature
(LCST) was 28.0[degree sign]C.The recovery of the polymer above the LCST was
over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human
serum system, and it has important functions in the human body. High purity HSA
is required in pharmaceuticals. Safe and efficient purification is a crucial
process during HSA production.
Results
A
thermo-response polymer was synthesized and L-thyroxin immobilized on the
polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST
of the affinity polymer was 31.0[degree sign]C and the recovery was 99.6% of
its original amount after recycling three times. The optimal adsorption
condition was 0.02 M Tris-HCl buffer (pH 7.0) and the HSA adsorption capacity
was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra
and a ForteBio Octet system were used to analyze the interactions between the affinity
polymer and HSA during adsorption and desorption. The recovery of total HSA by
elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied
to purification of HSA from human serum, HSA could be purified to single-band
purity according to SDS-PAGE.
Conclusion
A
thermo-response polymer was synthesized and L-thyroxin was attached to the
polymer. Affinity precipitation was used to purify HSA from human serum.
Tomado de envio
de bcm
No hay comentarios:
Publicar un comentario