## 2 Results and Discussion
### Products of the Recombinant LLA66  
LLA66 was first expressed with _E. coli_, but failed to yield a soluble protein. Then, the enzyme was overexpressed in a _S. cerevisiae_ INVSc1 strain, which is a fast-growing diploid strain ideal for expression by using a pYES2 vector that encodes a His-tag. The expression vector has the yeast _GAL1_ promoter for a high level of protein induction by galactose and repression by glucose, as well as the _URA3_ gene for selection of transformants in yeast host strains with a _ura3_ genotype. The His-tag was attached to the N or C terminus, but only C-terminally His-tagged LLA66 was soluble (Figure 3a). After the yeast was induced with 2% galactose, the cells were harvested and disrupted by a French press instrument. Immunoblotating against the recombinant LLA66 with a His-tag antibody revealed a molecular mass of 35.5 kDa band in the supernatant and in the Ni-NTA agarose-purified fractions (Figure 3b,c).  
Products of the purified recombinant LLA66 with FPP and IPP substrates were analyzed by HPLC. As shown in Figure 4a, the peak that eluted at 15.9 min corresponded to a C45 product under the same HPLC condition for analyzing the in vivo products of LLA66 in yeast. When the products where FPP and [14C]IPP were used as substrates were examined by TLC, a band corresponding to the C45 chain length was found (Figure 4b, lane 3), as compared to the C20-60 products generated by _E. coli_  
Figure 3: Purification of the recombinant LLA66. (**a**) Anti-His-tag western blot analysis of LLA66 linked with N- or C-terminal His-tag after different induction time periods. (**b**) SDS-PAGE analysis of the Ni-NTA purified LLA66. (**c**) Anti-His-tag western blot analysis of the Ni-NTA purified LLA66. M = markers, L = lysis supernatant, F = flow through, W = washed with 20 mM imidazole, and E = eluted with 300 mM imidazole.  
UPPS (lane 4) expressed in _E. coli_[17], consistent with the HPLC analysis. However, an extra band for C15 product was also detected (lane 3), even when only [14C]IPP was used as the substrate (lane 2). To investigate whether this C15 FPP was formed by LLA66 or yeast enzymes, we grew the yeast without LLA66 plasmid (pYES2-empty) and performed the same Ni-NTA chromatography. We found the fractions eluted by 300 mM imidazole contained the IPP-utilizing activity, and the product was C15 (lane 1). Therefore, this activity was due to an enzyme mixture binding to Ni-NTA. We further purified such an enzyme mixture through other columns (see below). To confirm that _S. cerevisiae_ is a suitable host to express long-chain _cis_-prenyltransferases, we also expressed human DHDDS and showed that the recombinant protein was active and produced expected long-chain products (lane 6) as compared to the C55 UPPS product generated in the presence of 0.1% Triton X-100 (lane 5).  
Figure 4: HPLC and TLC analysis of polyisoprenoids from recombinant LLA66 after Ni-NTA purification. **(a)** The C45 product extracted from the mixture of purified LLA66 with substrates FPP and IPP was eluted at 15.9 min under HPLC analysis. **(b)** C15 product was produced by using [14C]IPP and FPP with _S. cerevisiae_ cell lysate after Ni-NTA purification, as shown in lane 1. C15 and/or C45 products were generated by using [14C]IPP alone (lane 2) or [14C]IPP and FPP (lane 3) with _S. cerevisiae_ lysate expressing LLA66 after Ni-NTA purification. C20–60 products were produced by using [14C]IPP and FPP with the purified _E. coli_ UPPS in the absence of Triton X-100, as shown in lane 4. C55 was produced by using [14C]IPP and FPP with _E. coli_ UPPS in the presence of 0.1% Triton X-100, as shown in lane 5. Products with a variety of different chain lengths were generated by human DHDDS, as shown in lane 6.