Wet-milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine.
The production of recombinant proteins in plants continues to be of great interest for prospective large-scale manufacturing of industrial enzymes, nutrition products, and vaccines. This work describes fractionation by wet-milling of transgenic maize expressing the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B), a potent immunogen and candidate for oral vaccine and vaccine components. The LT-B gene was directed to express in seed by an endosperm specific promoter. Two steeping treatments, traditional steeping (TS, 0.2% SO(2) + 0.5% lactic acid) and water steeping (WS, water only), were evaluated to determine effects on recovery of functional LT-B in wet-milled fractions. The overall recovery of the LT-B protein from WS treatment was 1.5-fold greater than that from TS treatment. In both steeping types, LT-B was distributed similarly among the fractions, resulting in enrichment of functional LT-B in fine fiber, coarse fiber and pericarp fractions by concentration factors of 1.5 to 8 relative to the whole kernels on a per-mass basis. Combined with endosperm-specific expression and secretory pathway targeting, wet-milling enables enrichment of high-value recombinant proteins in low-value fractions, such as the fine fiber, and co-utilization of remaining fractions in alternative industrial applications.
Moeller
Lorena
L
Interdepartmental Plant Biology Major, Iowa State University, Ames, IA 50011, USA.
Taylor-Vokes
Raye
R
Fox
Steve
S
Gan
Qinglei
Q
Johnson
Lawrence
L
Wang
Kan
K
D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods*; D004768:Enterotoxins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D019344:Lactic Acid / Q000737:chemistry; D055100:Optical Fibers; D030821:Plants, Genetically Modified / Q000737:chemistry*; D012639:Seeds / Q000737:chemistry*; D013458:Sulfur Dioxide / Q000737:chemistry; D022462:Vaccines, Edible / Q000737:chemistry; D022223:Vaccines, Subunit / Q000737:chemistry / Q000302:isolation & purification*; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000302:isolation & purification*; D014867:Water; D003313:Zea mays / Q000737:chemistry*
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D022462:Vaccines, Edible; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D014867:Water; D013458:Sulfur Dioxide; D019344:Lactic Acid; C027538:heat-labile enterotoxin, E coli