Adrianus Marinus Ledeboer

CLONING OF CDNA ENCODING THE SWEET-TASTING PLANT PROTEIN THAUMATIN AND ITS EXPRESSION IN ESCHERICHIA COLI

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.

Synthesis and processing of the plant protein thaumatin in yeast

Various maturation forms of the plant protein thaumatin were expressed in yeast, using a promoter fragment of the glyceraldehyde- 3P -dehydrogenase (GAPDH) gene. Plasmids encoding preprothaumatin were shown to direct the synthesis of a processed form of the plant protein. The important role of signal sequences in the expression of the plant protein in yeast was indicated by the observation that plasmids encoding processed thaumatin forms were only poorly expressed, if at all. Nucleotide sequence analysis of the 843 nucleotide GAPDH promoter fragment revealed a characteristic structure with two regions of dyad symmetry containing translational starts of GAPDH and a putative 38 amino acid peptide. A promoter fragment from which the upstream region was deleted proved to be less efficient in thaumatin expression.

Cloning of the natural gene for the sweet-tasting plant protein thaumatin

Five different clones, homologous to the structural gene for the sweet-tasting plant protein thaumatin, have been isolated from leaf DNA of Thaumatococcus daniellii Benth. Restriction maps, hybridization studies, S1-nuclease mapping and R-loop formation revealed that the thaumatin genes isolated belong to one multigene family, and have two very small introns situated at different positions in the various structural genes. A similar situation prevails in a number of seed storage genes. This suggests a similarity between the sweet-tasting protein thaumatin and seed storage proteins.

Expression of cDNA encoding the sweet plant protein thaumatin in E. coli

tants it was shown that the transposon-specified site-specific recombination enzyme resolvase can replace the plasmid-encoded function of monomerizat ion of multimers. Using a series of Clo DF13 deletions as well as plasmids into which different parts of the Clo DF13 genome have been cloned, four regions, inc A, B, C and D could be identified that determine incompatibility (Hakkaar t et al., 1982b). The inc D region overlaps the replication control region of Clo DF13. The inc B region covers the Clo DF13 gene L. The gene L product, a protein with a molecular weight of 11 K D is also involved in plasmid stability and in the inhibition of multiplication of double-stranded phages like 2 and PI. In addition, two sites, inc A and ine C, are essential in the phenomenon of incompatibility. Site inc A is located in an AT-rich intercistronic region preceding the bacteriocine operon. The inc C site is situated in a region required for mobilisation of the plasmid, close to the origin o f transfer replication. The underlying mechanisms for stable maintenance of bacterial plasmids, e.g. for plasmid replication, replication control, stability and incompatibility are subject of current research in our laboratory.