No abstract

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag␣1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing ␣-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag␣1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of ␣-galactosidase-Ag␣1p was sixfold lower than that of ␣-galactosidase-Cwp2p and eightfold lower than that of ␣-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of ␣-galactosidase-Ag␣1p detectable. A lower translation efficiency of the ␣-galactosidase-AG␣1 fusion construct is most likely to be responsible for the low level of protein production. ␣-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.

No abstract

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).

The 271 nucleotides long scRNA (small cytoplasmic RNA) from Bacillus subtilis is structurally related to the Escherichia coli 4.5 S RNA (114 nucleotides), an essential molecule supposed to be involved in protein biosynthesis, but it possesses an additional moiety completely missing in the E. coli 4.5 S RNA. Both RNAs share a conserved hairpin with the eukaryotic 7SL RNAs, which mediate protein translocation as part of the signal recognition particle (SRP). We have cloned and sequenced the entire scRNA gene region from B. subtilis and have studied transcription and processing of the scRNA in B. subtilis by nuclease S1 mapping. This analysis revealed the scRNA gene to constitute a monofunctional transcription unit, expressed from a single promoter to a rho-independent terminator, yielding a precursor which extends the mature scRNA by approximately 40 nucleotides at both ends. Processing of the scRNA apparently involves only two endonucleolytic cuts and occurs first at the 5' end.