Expression, purification, and characterization of humanized anti-HBs Fab fragment.
Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.
Ning
Deng
D
Life Science and Technology College in Jinan University, Guangzhou, 510632, China. dengning123@sohu.com
Junjian
Xiang
X
Xunzhang
Wang
W
Wenyin
Chen
C
Qing
Zhang
Z
Kuanyuan
Su
S
Guirong
Rao
R
Xiangrong
Ren
R
Qingxin
Long
L
Zhouyao
Yu
Y
D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005822:Genetic Vectors; D006031:Glycosylation; D006510:Hepatitis B Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006510:Hepatitis B Antibodies; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins