(9a) Selective Recovery From Interstitial Spaces of Leaf Tissue

Title:  Selective Recovery of Apoplast-Targeted Recombinant Proteins from Plant Leaf Tissue

Nathanial J. Kingsbury and Karen A. McDonald

Department of Chemical Engineering and Materials Science

University of California, Davis, CA  95616

Transient production of recombinant proteins in plant leaf tissue using vacuum infiltration with recombinant Agrobacterium tumefacienshas advantages over other approaches including speed, scalability, safety and cost-effectiveness.  This method is being considered for large scale production of vaccines, biodefense agents and industrial enzymes and other products requiring a eukaryotic host. Recovery of functional protein from leaf tissue such as tobacco using traditional methods of homogenization and extractioncan be challenging due to high amounts of fiber, phenolics and proteases. Targeting  the product to the apoplast has advantages in terms of product quality, particularly glycoform type and uniformity, as well as ease of product recovery, since the secreted product can be removed from the leaf tissue without tissue disruption.  To recover transiently expressed apoplast-targeted recombinant proteins, a vacuum infiltration-centrifugation methodis applied to Nicotiana benthamiana and Nicotiana tabacum leaves.  Leaves are submerged in a buffer solution and a moderate vacuum is applied for several minutes and then quickly released, allowing the buffer solution to infiltrate the interstitial spaces of the leaf.  The leaf is then centrifuged to remove the liquid which is enriched with components from the apoplast and cell wall matrix. The collected fluid, called the apoplast wash fluid (AWF), is highly concentrated up to 16-fold and purified as much as 50-fold compared to a homogenate extract. Multiple rounds of vacuum infiltration-centrifugation, applied to further remove secreted protein from the residual tissue, can result in recovery ofover 90% of the secreted protein of interest.  The ratio of air phase to liquid phase in the interstitial space, called the dilution factor, is calculated and related to the rate of recovery from each round of vacuum infiltration-centrifugation.

Leaves subjected to the vacuum infiltration-centrifugation method showed no signs of damage after recovery of AWF and continued producing the recombinant protein of interest.  For example, when leaves expressing a recombinant cellulase enzyme, endoglucanase catalytic domain (E1cd) from Acidothermus cellulolyticus, were subjected to daily rounds of vacuum infiltration-centrifugation starting at two days post-agroinfiltration, the overall amount of E1cd collected fromthe leaves after six days post-agroinfiltration was as much as four times greater than in leaves where the  E1cd was allowed to accumulate in the apoplast unperturbed.  Possible mechanisms for this improvement in expression level are improved stability of the E1cd in the AWF compared to the leaf apoplast or improved secretion kinetics upon diluting the E1cd concentration in the apoplast and by extension the endomembrane system as a whole.  The results therefore demonstrate a novel technique for boosting expression levels in transgenic leaf tissue.

Out of the model proteins used these studies, those with smaller molecular weights were recovered with superior yields, suggesting that the porosity of the cell wall matrix may be limiting removal by the vacuum infiltration–centrifugation method.  Pretreatment of the leaves with low concentrations of pectinase enzyme improved the yield and concentration of an 82kDa recombinant protein, human butyrylcholinesterase, four-fold while improving its protein purity two-fold in the AWF.  These results demonstrate that even relatively large proteins can be efficiently recovered using the vacuum infiltration-centrifugation method.