title: Expression of Acidothermus cellulolyticus E1 endo-beta-1,4-glucanase catalytic domain in transplastomic tobacco.
abstract: As part of an effort to develop transgenic plants as a system for the production of lignocellulose-degrading enzymes, we evaluated the production of the endo-beta-1,4-glucanase E1 catalytic domain (E1cd) of Acidothermus cellulolyticus in transplastomic tobacco. In an attempt to increase the translation efficiency of the E1cd cassette, various lengths of the N-terminus of the psbA gene product were fused to the E1cd protein. The psbA gene of the plastid genome encodes the D1 polypeptide of photosystem II and is known to encode an efficiently translated mRNA. Experiments in an Escherichia coli expression system indicated that the fusion of short (10-22 amino acid) segments of D1 to E1cd resulted in modest increases in E1cd abundance and were compatible with E1cd activity. Plastid expression cassettes encoding unmodified E1cd and a 10-amino-acid D1 fusion (10nE1cd) were used to generate transplastomic tobacco plants. Expression of the E1cd open reading frame in transplastomic tobacco resulted in very low levels of the enzyme. The transplastomic plants accumulated a high level of E1cd mRNA, however, indicating that post-transcriptional processes were probably limiting the production of recombinant protein. The accumulation of 10nE1cd in transplastomic tobacco was approximately 200-fold higher than that of unmodified E1cd, yielding 10nE1cd in excess of 12% of total soluble protein in the extracts of the lower leaves. Most importantly, the active recombinant enzyme was recovered very easily and efficiently from dried plant material and constituted as much as 0.3% of the dry weight of leaf tissue.
authors.affiliation: Biotechnology Center, University of Wisconsin-Madison, 425 Henry Mall, Madison, WI 53706, USA. t_zieg@biotech.wisc.edu
authors.forename: Thomas
authors.initials: T
authors.lastname: Ziegelhoffer
authors.forename: John A
authors.initials: JA
authors.lastname: Raasch
authors.forename: Sandra
authors.initials: S
authors.lastname: Austin-Phillips
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D016366:Open Reading Frames; D045332:Photosystem II Protein Complex / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D018087:Plastids; D012333:RNA, Messenger / Q000378:metabolism; D018749:RNA, Plant / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D045332:Photosystem II Protein Complex; D012333:RNA, Messenger; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; C422369:photosystem II, psbA subunit; D002480:Cellulase