Expression and secretion vectors for hirudine by way of transformed yeasts

Claim 2

Transformed yeast according to Claim 1, characterized in that the said functional block contains at least the following sequences, from the 5' end to the 3' end: - a sequence (S tr ) containing the signals which provide for transcription of the H gene by yeast, - a leader sequence (L ex ) containing the elements required for obtaining excretion of the gene product, - the gene coding for hirudin, one of its variants or a precursor of these, - the gene coding for hirudin or one of its variants or precursors of these, preceded by a cleavage site S cl .

Claim 3

Yeast according to either of Claims 1 and 2, characterized in that the sequence L ex is that of the alpha sex pheromone of yeast.

Claim 4

Yeast according to one of Claims 1 to 3, characterized in that the leader sequence contains the pre fragment of the alpha sex pheromone of yeast.

Claim 5

Yeast according to one of Claims 1 to 4, characterized in that the block contains the prepro sequence of the alpha sex pheromone of yeast at the 5' end of the H gene.

Claim 6

Yeast according to one of Claims 1 to 5, characterized in that the H gene is followed by a yeast terminator sequence.

Claim 7

Yeast according to Claim 6, characterized in that the yeast terminator sequence is that of the PGK gene.

Claim 8

Yeast according to one of Claims 1 to 7, characterized in that the cleavage site is the dipeptide Lys-Arg.

Claim 9

Yeast according to one of Claims 1 to 7, characterized in that the functional block is integrated in a plasmid containing at least one origin of replication in yeasts.

Claim 10

Yeast according to Claim 9, characterized in that the origin of replication is that of the 2» plasmid.

Claim 11

Yeast according to one of Claims 9 and 10, characterized in that the plasmid contains, in addition, a selectable character.

Claim 12

Yeast according to Claim 11, characterized in that the selectable character is given by the URA3 gene.

Claim 13

Yeast according to one of Claims 1 to 12, characterized in that it is a yeast of the genus Saccharomyces .

Claim 14

Yeast according to Claim 13, characterized in that it possesses the Matalpha mating type.

Claim 15

Yeast according to one of Claims 12 to 14, characterized in that it is a strain of S. cerevisiae .

Claim 16

Process for preparing hirudin by fermentation of a yeast according to one of Claims 1 to 15 in a culture medium and recovery of the hirudin produced in the culture medium.

Claim 17

Process for preparing hirudin by fermentation of a yeast, characterized in that: a) a yeast strain containing a functional DNA block carried by a plasmid or integrated in a chromosome of the said yeast is cultured in a culture medium, characterized in that the DNA block contains at least the sequence: ---S tr ---L ex ---S cl ---H gene---ter · S tr is a DNA sequence containing the signals which provide for transcription of the H gene by yeast, · L ex is a leader sequence enabling the excretion of hirudin, mature or in the form of a hirudin precursor which can be matured in vitro, to be obtained, · S cl is a DNA signal coding for a cleavage site chosen from ATG and the sequences coding for Lys-Arg; b) the hirudin produced is recovered in the culture medium in mature form or in the form of a hirudin precursor which can be matured in vitro.

Claim 18

Process according to Claim 17, characterized in that the sequence S cl -H gene contains a sequence coding for the sequence Lys-Arg-Ile-Thr.

Claim 19

Extracorporeal blood circuit, characterized in that at least part of the circuit in contact with the blood possesses a neutral surface of hirudin obtained by a process according to one of Claims 16 to 18.

Claim 20

Anticoagulant composition, characterized in that it contains yeasts according to one of Claims 1 to 15 as an agent which liberates hirudin.

Claim 21

Composition for visualizing the formation of a blood clot, characterized in that it consists of labelled hirudin obtained by a process according to one of Claims 16 to 18.