Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

<p>Abstract</p> <p>Background</p> <p>Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in <it>E. coli </it>had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in <it>E. coli</it>.</p> <p>Results</p> <p>An expression plasmid containing the open reading frame for tung tree (<it>Vernicia fordii</it>) DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in <it>E. coli </it>BL21(DE3). Immunoblotting showed that the recombinant DGAT1 (rDGAT1) was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification.</p> <p>Conclusions</p> <p>This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.</p

Oil accumulation in developing tung seeds.

<p>Lipids were extracted from developing tung seeds collected weekly. Total seed oil content was determined by weight. Seed lipid composition was determined by gas chromatography after converting all lipids to methyl esters. The peaks corresponding to lipid fatty acids were identified by comparing to residence times for fatty acid methyl ester standards.</p

Tung tree DGAT amino acid sequence alignment.

<p>The full-length protein sequences of tung DGAT1, DGAT2 and DGAT3 were aligned using the ClustalW algorithm of the AlignX program of the Vector NTI software. The nucleotide sequence alignment of tung tree DGATs is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076946#pone.0076946.s001" target="_blank">Figure S1</a>.</p

Relative expression of DGAT genes in tung seeds, leaves and flowers.

<p>TaqMan qPCR reaction mixtures contained variable amounts of RNA-equivalent cDNAs (2.5, 5, 12.5 and 25 ng) from tung tissues and the optimized concentrations of each primer and probe (200 nM). The mean and standard deviation of mRNA expression levels calculated from the four concentrations of DNA templates are presented. The results are representative data from three experiments. (A) Rpl19b as the reference mRNA. (B) Gapdh as the reference mRNA. (C) Ubl as the reference mRNA. T1 represents RNA extracted from tung tree 1. The number after T1 represents the week when tung seeds were collected.</p

Northern blotting of DGAT1, DGAT2 and DGAT3 mRNA in tung tree tissues.

<p>Total RNA was extracted from tung leaves, flowers and developing seeds. Total RNA (15 µg RNA/lane) was blotted and probed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076946#s2" target="_blank">Materials and Methods</a>”. The DGAT1, DGAT2 and DGAT3 blots were developed for 10 min, 20 min and 50 min, respectively. Labels above the panels: 1–11-RNA isolated from tung seeds collected at week 1–11; L-RNA from leaves; Fl-RNA from flowers.</p

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

<div><p>Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (<i>Vernicia fordii</i>), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.</p></div

Phylogenetic analysis and identification of amino acid residues and sequence motifs conserved in DGAT3s.

<p>(A) Phylogenetic analysis. The presumed evolutionary relationships among the 27 DGATs from 19 organisms were analyzed by phylogenetic analysis. The numbers in the parenthesis following DGAT names are the calculated distance values, which reflect the degree of divergence between all pairs of DGAT sequences analyzed. (B) Identification of amino acid residues and sequence motifs conserved in DGAT3s. Multiple sequence alignment was performed using the ClustalW algorithm of the AlignX program of the Vector NTI software. Each DGAT sequence name is on the left of the alignment followed by the position of amino acid residue of DGAT protein sequence in the alignment. The numbers before, in the middle and after the amino acid residues in the sequence alignment represent the number of residues in the divergent region as previously used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076946#pone.0076946-Cao10" target="_blank">[63]</a>. The letters at the bottom of the alignment are the consensus residues. Color codes for amino acid residues are as follows: 1) red on yellow: consensus residue derived from a completely conserved residue at a given position; 2) blue on cyan: consensus residue derived from the occurrence of greater than 50% of a single residue at a given position; 3) black on white: non-similar residues. The complete sequence alignment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076946#pone.0076946.s002" target="_blank">Figure S2</a>. The abbreviations of the organisms are: Ah, <i>Arachis hypogaea</i> (peanut); At, <i>Arabidopsis thaliana</i>; Bd, <i>Brachypodium distachyon;</i> Gm, <i>Glycine max</i> (soybean); Hv, <i>Hordeum vulgare (barley)</i>; Lj, <i>Lotus japonicas</i>; Mt, <i>Medicago truncatula</i>; Os, <i>Oryza sativa</i> (rice); Pg, <i>Picea glauca</i> (white spruce); Pp, <i>Physcomitrella patens</i>; Ps, <i>Picea sitchensis</i> (sitka spruce); Pt, <i>Populus trichocarpa</i>; Rc, <i>Ricinus communis</i> (caster bean); Sb, <i>Sorghum bicolor</i> (sorghum); Sl, <i>Solanum lycopersicum</i> (tomato); Sm, <i>Selaginella moellendorffii</i>; Vf, <i>Vernicia fordii</i> (tung tree); Vv, <i>Vitis vinifera</i> (grape); Zm, <i>Zea mays</i> (corn).</p

The properties and amino acid composition of the tung DGAT family and DGAT3 subfamily with 27 DGATs from 19 plant species.

<p>The properties and amino acid composition of the tung DGAT family and DGAT3 subfamily with 27 DGATs from 19 plant species.</p