Chromatographic enrichment of jojoba FAR activity. A, Blue A agarose chromatography. Solubilized jojoba microsomal membranes were applied to a Blue A agarose column in a buffer containing 0.4 m NaCl. FAR activity was eluted using a buffer containing 1.0 m NaCl. FAR activity is shown as picomoles of alcohol formed per minute per fraction. Relative protein concentrations of the various fractions are shown as the A₂₈₀ value of the UV monitor. The horizontal bar indicates fractions pooled for subsequent chromatography. Arrows indicate when buffer solutions containing other than 0.4 m NaCl were applied to the column. B, Sephacryl S-100 chromatography. Pooled fractions from the Blue A agarose column were concentrated and applied to the column. Activity, relative protein, and fractions pooled for the next chromatographic step are indicated as in A. C, Palmitoyl-CoA agarose chromatography. Pooled fraction from the size exclusion column step were applied to a palmitoyl-CoA-agarose column. After washing with equilibration buffer and then buffer containing NADH, FAR activity was eluted with buffer containing NADPH. The effect of subsequent washing with a buffer containing 0.5 m NaCl is also shown. Arrows indicate when various solutions were applied to the column.

SDS-PAGE showing purification of jojoba FAR. Proteins were resolved on a 10% to 15% polyacrylamide gradient gel and stained with silver (Blum et al., 1987). The samples loaded onto the gel were taken from a representative purification sequence. Lane 1, Cell-free homogenate; lane 2, microsomal membranes; lane 3, detergent-solubilized 200,000g supernatant fraction; lane 4, Blue A agarose column (1.0 m NaCl elution); lane 5, size-exclusion column, pooled retained fractions; lane 6, palmitoyl-CoA agarose column (NADPH elution); and lane 7, molecular mass standards.

Amino acid sequence of the jojoba FAR. The predicted amino acid sequence of the jojoba FAR protein was translated from the cDNA. Tryptic peptide sequences derived from both the 54- and 56-kD proteins are double-underlined. Tryptic peptide sequences derived only from the 56-kD protein are single-underlined. A sequence derived from a cyanogen bromide peptide is shown as an overline. The N-terminal sequence of a 32-kD protein used to design a primer to isolate the cDNA clone is underlined with a dashed line. Two potential transmembrane helixes are shaded.

TLC analysis of E. coli lipids. Lipids were extracted from E. coli, analyzed using normal phase TLC, and visualized by staining with iodine. The standards lane contains a wax ester (jojoba oil), a free fatty acid (oleic acid), and a fatty alcohol (oleyl alcohol). The lane labeled pET3A contains lipids from E. coli transformed with the empty expression vector pET3A. The lane labeled pCGN7800 contains lipids from E. coli transformed with the pET3A containing the jojoba FAR.

Fatty alcohols and wax esters in transgenic HEAR seed oils. A, Alcohol content of the pooled seeds from plants transformed with the native and resynthesized FAR genes. Pooled seeds from transgenic plants containing either the native jojoba FAR or the synthetic gene designed to reduce AT content were analyzed by transmethylation of the seed oil and GC. The frequency of independent transgenic plants whose seed alcohol content fell in the various ranges was plotted against the amount of fatty alcohol present in the seed oil. Gene resynthesis increased both the frequency of transgenic plants with detectable fatty alcohol in their oil and the quantity of fatty alcohol found in the seed oil. B, Wax versus alcohol content of individual seeds. The wax ester and fatty alcohol content of single seeds from transgenic plants containing the synthetic FAR were determined as described in “Materials and Methods.” The upper line represents complete esterification of the fatty alcohol into wax esters, and the lower line represents 50% esterification of the fatty alcohols into wax esters. As the fatty alcohol content of the seed increases, the endogenous wax synthase fails to esterify all of the fatty alcohol into wax esters.

A + T content of the native jojoba FAR open reading frame (ORF) and a synthetic ORF. The A + T content of 25 nucleotide windows of the jojoba FAR ORFs were plotted against their position in the ORFs. A, The A + T content of the native jojoba FAR ORF. Several regions of the ORF have A + T contents exceeding 75%. B, The A + T content of a synthetic FAR ORF designed to reduce the A + T content without changing the protein encoded by the native FAR ORF.