It is possible as a consequence of such comparative study for particularly efficient signal sequences to be identified, cloned and used in all three possible translation reading frames for the expression of heterologous proteins.It is possible in principle to distinguish between two different types of signal sequences: a "hydrophobic" type and a "hydrophilic" type.

Xxx coli-31

The invention relates to a new signal peptide from Bordetella pertussis with the amino acid sequence M K K W F V A A G I G A G L L M L S S A A and to particularly suitable expression vectors with whose aid such signal sequences can be found and/or evaluated.

The invention relates to the signal peptide of a protein from Bordetella pertussis which is able to direct heterologous proteins into the periplasmic space between the inner and outer membranes of Gram-negative species of bacteria.

The transport of a heterologous protein into the periplasmic space in E. USA (1985) 82, 8129-8133) continued this work by placing the c DNA coding for Pho A without a signal sequence and 5 subsequent amino acids on the 3' side in front of the transposon Tn5 (loc. Bacteriology (1988) 170, 5059-5066) have now generated, by means of Tn Pho A mutagenesis, mutants in Bordetella pertussis which are influenced by modulation signals (in this case nicotinic acid and Mg SO), with the majority of these mutants being repressed and some being activated, which suggests that there are at least two trans-acting regulatory genes.

coli substantially corresponds functionally to the transport of a protein into the lumen of the endoplasmic reticulum of eukaryotic cells. USA; (1985) 82, 5107-5111) describe plasmids which code for the periplasmic alkaline phosphatase from E. cit.) and were thus able to show that fusions not only with secreted proteins but also with membrane proteins result in active Pho A. We have found that the mutant SK6 mentioned therein contains a new and very efficient signal sequence. Furthermore, the invention is further detailed in the examples and the patent claims. coli Pho A structural gene derivative which has no signal sequence.

It is composed of a relatively hydrophilic NH terminus with one or two basic amino acids, of an apolar, mostly hydrophobic block of seven or eight amino acids, and of a relatively hydrophilic COOH terminus which is terminated by an amino acid with a small side-chain.

Such "hydrophobic" signal sequences guide proteins through the membrane of the endoplasmic reticulum (ER) and through bacterial membranes.The deletion derivatives obtained in this way were recloned into the plasmid p LAFR2 which is able to replicate in B. (1982), Gene 18, 289-196) and, after conjugative transfer into B.pertussis, examined for Pho A activity susceptible to modulation.In this way a Pst I fragment which is about 3.2 kb in size was identified and subcloned into p UC18 (called p UC-PI hereinafter) which now contains only about 500 base-pairs of B. pertussis derivatives which contain the cloned Bam HI fragment which is 20 kb in size or the Pst I fragment which is 3.2 kb in size do not differ essentially in their phosphatase activity, the transcriptional and translational regulation sequences of the vrg6 gene fusion on the latter fragment must still be completely present. pertussis signal sequence characterized in this way comprises 21 amino acids and was subsequently prepared and cloned as described in Example 3 and is suitable for the secretion of heterologous proteins. pertussis-specific nucleotides upstream from the Tnpho A insertion site, followed by a p UC18-specified Sac I cleavage site (Tab. Pst I/Sac I cleavage of the vrg6-delta11 DNA results in the complete Pho A structural gene from Tnpho A, which has no signal sequence and is on a fragment which is about 2.6 kb in size and which serves as a source of the pho A structural gene which has no signal sequence in Example 2.pertussis DNA upstream of the Tnpho A insertion site of the vrg6 gene fusion and is Pho A positive in B. Starting from p UC-PI, deletions were introduced into the DNA region located 500 base-pairs upstream from the Tnpho A insertion site using the enzymes exonuclease III and S1 nuclease by the method of Henikoff ((1984) Gene 28, 351-359). Construction of a vector plasmid (p Trc99C-pho A) for the cloning and comparative efficiency measurement of signal sequences.The "hydrophobic" group of signal sequences usually comprises about 13-30 amino acids, whereas the "hydrophilic" group comprises about 12-70 amino acids.