Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells.
Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.
Huang
J
J
Applied Phytologics, Inc., 4110 North Freeway Boulevard, Sacramento, CA 95834, Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-Cho, Sakai 599-8531, Japan. JHuang@APINC.com
Sutliff
T D
TD
Wu
L
L
Nandi
S
S
Benge
K
K
Terashima
M
M
Ralston
A H
AH
Drohan
W
W
Huang
N
N
Rodriguez
R L
RL
D000818:Animals; D019470:Biolistics; D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D006801:Humans; D059828:Plant Cells; D010944:Plants / Q000378:metabolism*; D030821:Plants, Genetically Modified; D051381:Rats; D017207:Rats, Sprague-Dawley; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics
D000515:alpha 1-Antitrypsin