BGAF was extracted four times from 8 g of 3-d-old H95 etiolated shoots in 50 mM NaAc buffer, pH 5.0, containing 30 % ammonium sulfate (AS). The final pellet containing primarily free BGAF was solubilized in 50 mM NaAc buffer, pH 5.0 and immediately applied to a gel filtration column (Sephacryl HR 200, 90cm x 16mm). Fractions containing BGAF were identified by ELISA and pooled for hydrophobic interaction chromatography. BGAF was then applied to a ToyoPearl-butyl 650M hydrophobic interaction chromatography at 0.8 M AS in 50 mM NaAc buffer, pH 5. The column was washed with 0.8 M AS in NaAc buffer until baseline was reached. Stepwise elution was performed by reducing the ammonium sulfate concentration in 0.1 M increments. Fractions were screened for BGAF by ELISA, pooled, and concentrated 12-fold on a 10 K cut-off spin column (Gelman Sciences).
Purified BGAF (~250 pmoles) was submitted to Commonwealth Biotechnology for N-terminal sequencing. The N-terminal sequence was identified to be: [V, ?] [I, E] [G, P] [N, L] YAPIGIGATV. Therefore, the peptide APIGIGAT was used to design two degenerate primers; BGAF-6, CCNATHGGNATHGGNGCNAC; BGAF-7, CNCCNATHGGNATHGGNGC.
H95 seedlings were germinated in the dark for 2 days at 30 C in wet vermiculate. Whole shoots were harvested with a razor blade and used immediately for RNA isolation. mRNA was isolated using oligo dT coated magnetic beads. An Oligo dT primer (RT-3) was used for first strand cDNA synthesis with AMV-reverse transcriptase. To amplify the BGAF cDNA, BGAF-6 and BGAF-7 were individually paired with RT-3 in separate PCR reactions. The 1 kb PCR product generated was reamplified with pfu Turbo DNA polymerase, gel purified, and blunt-end cloned into pBluescript" II SK (+/-) for sequencing in both directions.
The sequence was used to perform a BLAST search in the maize EST database. The BLAST search exclusively identified heat-shock proteins, which matched with similarities ranging from 56% - 96%. The extreme 5' end of BGAF was obtained by overlapping the BGAF sequence with the highest match (96%) in the EST database. The identity of the 5' end sequence obtained by overlap was corroborated by reamplification of the H95 cDNA using a primer designed from the extreme 5' end of the EST sequence paired with a BGAF specific 3' end primer.
The identity of the putative BGAF cDNA was shown following its expression in E. coli. Western blots of E. coli expression extracts probed with BGAF antiserum showed an immunoreactive band whose electrophoretic mobility and molecular size were identical to BGAF isolated from plant extracts. Additionally, functional assays clearly showed the presence of BGAF in E. coli expression extracts, which tested positive for b-glucosidase binding activity in gel-shift assays.
BGAF has a 1143-bp long cDNA sequence, which includes a 918-bp coding sequence, and a 172-bp 3' untranslated region. The incomplete 5’ untranslated region is 53-bp long.
The deduced protein has an open reading
frame (the 918 bp coding region) encoding a 306- amino acid-long mature
protein with a calculated molecular mass of 31.8 kD (Fig. 1). BGAF has
a calculated isoelectric point of 6.00, consistent with the experimental
1. Deduced primary structure of the BGAF cDNA. The open reading frame
of BGAF encodes a 306-amino acid-long mature protein. The N-terminal protein
sequence from which degenerate primers for RT-PCR were designed (arrow,
gray box) is shown. The fact that the N-terminal sequence obtained is an
internal polypeptide is likely due to an endogenous protease in maize.
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