(i) Choice of Expression System:
     Because the majority of our target hydrolases are likely to be glycosylated, we chose Pichia pastoris as the primary system for heterologous expression. This system offers many advantages, including ease of use, high expression levels, and the ability to execute typical eukaryotic posttranslational modifications. 

(ii) Obtaining cDNAs Encoding Mature Proteins:
     Although we initially expected to obtain a substantial number of full-length ß-glucosidase and ß-galactosidase cDNAs from clone banks and individual laboratories, this proved incorrect. Of the 21 ß-glucosidase genes assigned to UI, clones for only three (Glu6, Glu8, and Glu13) were available. Furthermore, we learned that the 14 ß-galactosidase cDNA sequences submitted to GenBank by Gy et al. (1999) were contigs derived from sequencing overlapping PCR fragments (Gy et al., personal communication). Thus, apart from the AtBGAL17 cDNA (AY058198), no other full-length ß-galactosidase cDNA clones were available from ABRC or other sources.

    With the four exceptions noted above, cDNAs encoding the mature regions of all ß-glucosidase and ß-galactosidase proteins assigned to UI are being obtained by PCR amplification of full-length cDNA populations. To this end, total RNA was isolated from Arabidopsis leaves, roots, flowers, and siliques using the Trizol reagent. Poly(A)⁺ RNA was then obtained using the Oligotex Kit (Qiagen). cDNA populations enriched for full-length clones were synthesized from poly(A)⁺ RNA by a modification of the SMART-cDNA Synthesis procedure (Clontech), in which Improm-II reverse transcriptase (Promega) was used. Subsequent amplification was undertaken using gene-specific primers.

(iii) Cloning Strategies:
     To clone target genes, RT-PCR was undertaken with Takara Ex Taq polymerase (or Clontech Advantage-HF 2) using primers that incorporate specific restriction sites. Taking advantage of the extra A added at the 3'-ends by these polymerases, we then used TA-cloning to ligate the PCR product (after purification, if necessary) into the pCR4-TOPO vector (Invitrogen). (In some cases, the pCR2.1-TOPO vector served as intermediate vector). Double-strand sequencing of the insert was carried out at this stage to: (a) confirm that the deduced amino acid sequence of the desired hydrolase, as published by GenBank, is correct, and (b) ensure that no unintended mutations had been introduced by PCR. The insert was then excised and cloned directionally into the pPICZaB vector (Invitrogen) in frame with the Saccharomyces cerevisiae a-factor signal sequence, which allows for efficient secretion of the recombinant hydrolase. The resulting plasmid was transformed into competent Pichia cells by electroporation or chemical methods.

     Several variations on this cloning strategy are currently being tested. First, to reduce the undesired mutations observed in Taq polymerase-generated PCR products, we have amended this strategy to allow use of the proofreading polymerase KOD HiFi DNA polymerase (Novagen). After PCR, the blunt-ended PCR products are A-tailed by brief incubation with Taq polymerase, prior to TA-cloning into the pCR-TOPO vector. In the foregoing approaches, PCR primers were designed so that the stop codon of the target protein is retained. In some cases, however, we have cloned the PCR product into the Pichia vector in frame with C-terminal myc epitope and polyhistidine tags. The latter will facilitate detection and purification of tagged recombinant hydrolases.