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Expression and Purification of Functional Human α‐1‐Antitrypsin from Cultured Plant Cells
Author(s) -
Huang Jianmin,
Sutliff Thomas D.,
Wu Liying,
Nandi Somen,
Benge Kelli,
Terashima Masaaki,
Ralston Annemarie H.,
Drohan William,
Huang Ning,
Rodriguez Raymond L.
Publication year - 2001
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0001516
Subject(s) - biology , cloning (programming) , microbiology and biotechnology , gene , transformation (genetics) , cell culture , elastase , protease , gene expression , signal peptide , expression vector , enzyme , biochemistry , recombinant dna , genetics , computer science , programming language
Human α‐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 RAmy 3D 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.