For this purpose, the Bam HI fragment which is 20 kb in size was subjected to restriction analysis, and subfragments which carry the entire Pho A sequence from Tnpho A but, compared to the 20 kb fragment, truncated B.

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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.

Hence the object was to isolate particularly efficient signal sequences and to design processes suitable for this. Accordingly, the invention relates to: a) the signal sequence ##STR2## b) plasmids which carry a sequence of this type, c) the use thereof for the secretion of proteins, and d) plasmids which are particularly suitable for the cloning and quantitative evaluation of signal sequences, due to the fact that a strong promoter which can be regulated, such as trc, is followed by the lac Z ribosome-binding site (RBS) and by a vector-encoded translation initiation codon at a distance from the lac Z RBS which is optimized for high expression, with an Nco I cleavage site being present directly at the 5' end of the Pho A structural gene which has no signal sequence, but having been deleted from within the Pho A sequence by mutation, and with p Trc99C-Pho A being preferred. 1 (Parts a and b): Construction of plasmid p Trc99C-pho A. coli Pho A structural gene from Tn Pho A and the signal sequence of the structural gene affected by the transposition coincide.

It is easy to identify such Pho A positive colonies using the dyestuff indicator 5-bromo-4-chloro-indoxyl phosphate toluidine (XP).

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.

The single strand with the cloned Eco RI fragment obtained in this way was then isolated by known methods and subjected to the published gapped-duplex mutagenesis protocol (Kramer et al. Subsequently the Eco RI fragment which is 330 base-pairs in size was reisolated from this plasmid and sited in place of the corresponding fragment of the plasmid pvrg-6-delta11.

For this purpose, pvrg-6-delta11 was partially digested with Eco RI, and a fragment which was shorter by 330 base-pairs than the starting plasmid pvrg-deltall (about 6700 bp), which had been linearized by partial Eco RI digestion, was isolated.

The pho A structural gene carries an internal Nco I cleavage site.