Method for exposing peptides and polypeptides on the cell surface of bacteria

• US 20040106118A1
• Filed: 10/15/2003
• Published: 06/03/2004
• Est. Priority Date: 10/26/2000
• Status: Active Grant

• Current Assignee: Biontech AG
• Original Assignee: Nascacell Technologies AG
• Inventors: Kolmar, Harald, Christmann, Andreas, Wentzel, Alexander
• Granted Patent: US 7,186,524 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 11/16 Enzymes or microbial cells immobilised on or in a biological cell
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

The inventive method allows peptides or polypeptides to be exposed on the surface of gram-negative host bacteria using specific intimin-based anchor modules. Intimins with shortened carboxy terminals have been found to be particularly suitable anchor modules for passenger domains in the exterior E.coli cell membrane. According to said method, host bacteria are transformed using vectors, on which are located a fused nucleic acid sequence consisting of a sequence segment which codes for an intimin with a shortened carboxy terminal and a nucleic acid sequence segment which codes for the passenger peptide that is to be exposed. The invention permits a particularly large number of passenger domains to be exposed on the cell surface of the bacteria, without adversely affecting the viability of the bacteria.

Claims

1. Process for exposing peptides and proteins on the surface of host bacteria, in which one

(a) prepares a Gram-negative host bacterium that is transformed with a vector on which a fused nucleic acid sequence is localized which is in operative linkage with an exogenously inducible promoter that

(i) codes for a sequence segment which is an Intimin shortened by at least the C3 domain in the carboxyterminal region as the anchoring domain, and

(ii) has a nucleic acid sequence segment coding for the passenger peptide or passenger polypeptide to be exposed, and

(b) cultivates the host bacterium under conditions in which the peptide/polypeptide/protein coded by the nucleic acid segment (ii) is expressed and exposed on the surface of the host bacterium, whereby the nucleic acid sequence segment (ii) is heterologous with respect to the nucleic acid sequence segment coding for the Intimin membrane anchoring domain.

2. Process according to claim 1,

characterized in that the shortened Intimin is shortened in the carboxy terminal region of 280 amino acids by at least one other of the Intimin domains D0, D1, D2.

3. Process according to claim 1,

characterized in that

the Intimin anchoring domain used in the external bacterial membrane was derived from the genus [sic/tr. note #10] of the Enterobacteriaceae and is used in a host bacterium of a genus of the Enterobacteriaceae.

4. Process according to claim 3,

characterized in that

a fragment of the Intimin from enterohemorrhagic E. coli or a variant of it is used as the anchoring domain.

5. Process according to claim 4,

characterized in that

a carboxy-terminal shortened variant of the Eae Intimin from hemorrhagic Escherichia coli O157:

H7 is used as the anchoring domain.

6. Process according to claim 5,

characterized in that

the shortened variant of the Eae Intimin contains the amino acids 1 to 659.

7. Process according to claim 5,

characterized in that

the shortened variant of the Eae Intimin contains the amino acids 1 to 753.

8. Process according to claim 1, in which the expression of the fusion gene from the nucleic acid sequence segment coding for the shortened Intimin and the nucleic acid sequence segment coding for the passenger protein can be controlled, whereby

(i) a codon of the nucleic acid sequence coding for the shortened Intimin which codes for glutamine is replaced by an amber stop codon (TAG), and

(ii) an Escherichia coli host strain is used in which translation of the mRNA of the fusion gene is accomplished by providing a controllable amount of suppressor tRNA which allows over-reading of the stop codon in the translation.

9. Process according to claim 8, in which the regulation of the expression of the gene for the amber suppressor tRNA takes place due to the fact that it is placed under control of a promoter which is controllable in its transcription rate.

10. Process according to claim 9, in which the promoter, which is controllable in its transcription rate, is the PLlac promoter.

11. Process for producing a variant population of surface-exposed peptides/polypeptides/proteins and for identifying bacteria, which carry the peptides/polypeptides/proteins with a desired property, in which the process has the following steps:

(i) production of one or more fusion genes or fusion gene fragments by cloning the coding sequence of a desired passenger in the continuous reading frame with the coding sequence of the shortened Intimin gene in at least one expression vector;

(ii) variation of the passenger peptide by one of the methods selected from;

intentional site-directed mutagenesis, e. g., through the polymerase chain reaction (PCR) using oligonucleotides with intentionally exchanged bases, by random mutagenesis using oligonucleotides with randomly generated base sequences in selected sequence segments in the PCR, through error-prone PCR, randomly controlled chemical or radiation-generated mutagenesis,

(iii) introducing at least one vector into the host bacteria which expose the passenger stably on their surfaces,

(iv) expression of the fusion gene in the host bacteria,

(v) cultivation of the bacteria to produce a stable surface-exposed passenger.

12. Process according to claim 11, which further comprises:

(vi) selective enrichment of the bacteria which carrier the passenger with the desired properties on the surface.

13. Process according to claims 11 or 12, in which the identification of the bacterium which carries a passenger with a desired binding affinity exposed on the surface is accomplished by binding to an immobilized and/or labeled binding partner.

14. Process according to claim 11 or 12, in which a population of bacteria which carry a surface-exposed passenger with a binding affinity to a binding partner is used as the matrix for affinity chromatography purification of the binding partners from a mixture of substances.

15. Expression vector for the expression of a fusion gene under the control of an exogeneously inducible promoter in which there is, in operative linkage with the promoter, a coding sequence for a desired passenger peptide in the continuous reading frame downstream to a coding sequence for an Intimin shortened by at least the D3 domain in the carboxy-terminal region of 280 amino acids s the anchoring domain for the passenger peptide

16. Expression vector according to claim 15, in which the promoter is selected from the group lac promoter, ara promoter, tetA promoter.

17. Gram-negative host bacterium of the genus [sic/tr. note #10] Enterobactericeae, transformed with at least one expression vector according to claim 15.