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Expression and secretion of heterologous polypeptides from caulobacter

a technology of heterologous polypeptides and caulobacter, which is applied in the field of expression and secretion of heterologous polypeptides from caulobacter, can solve the problems of limited opportunities limited nutrient availability, and limited use of such surface proteins as a vehicle for expression and/or presentation of heterologous polypeptides, etc., to promote secretion and culture rapid

Inactive Publication Date: 2002-01-24
RES CORP TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0029] A heterologous polypeptide referred to herein may be any peptide, polypeptide, protein or a part of a protein which is desired to be expressed in Caulobacter and which may be secreted by the bacterium. The heterologous polypeptide includes enzymes and other functional sequences of amino acids as well as ligands, antigens, antigenic epitopes and haptens. The size of the heterologous polypeptide will be selected depending upon whether an intact S-layer is to be produced in the Caulobacter or whether the chimeric protein to be recovered from the bacterial medium as described below. Preferably, the cysteine content of the heterologous polypeptide and the capacity for formation of disulphide bonds within the chimeric protein will be kept to a minimum to minimize disruption of the secretion of the chimeric protein.
[0058] This invention also provides an efficient expression system for polypeptides that may be harvested in large quantities relatively free of contaminants and protein of Caulobacter origin. Expression of a heterologous polypeptide fused with sufficient C-terminal amino acids of the rsaA protein to promote secretion of the heterologous polypeptide results in the accumulation of large quantities of secreted protein in the cell medium. In such cases, the chimeric protein does not have to be released from the cell surface. Furthermore, adjustment of the size of the N-terminal rsaA portion can dictate whether the secreted protein is soluble or will precipitate in the cell medium. This embodiment may also be useful in cases where the Caulobacter is to express a foreign antigenic component and it is desired to minimize the amount of Caulobacter protein that is associated with the foreign antigen secreted by the Caulobacter.

Problems solved by technology

Generally, the use of such surface proteins as a vehicle for expression and / or presentation of heterologous polypeptides has been limited by the characteristics of a particular surface protein.
Where the surface protein is functional (for example, as part of a filamentous portion of a bacterial cell surface) there will be limited opportunities to express a fusion product and still retain the surface protein's function.
This nutrient can be limiting in many leachate waste streams, especially those with high levels of iron or calcium.

Method used

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  • Expression and secretion of heterologous polypeptides from caulobacter
  • Expression and secretion of heterologous polypeptides from caulobacter
  • Expression and secretion of heterologous polypeptides from caulobacter

Examples

Experimental program
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example 2

Insertion of Cadmium Binding Polypeptides into Specific Sites

[0061] An insertion of the above described 12 bp linker was made at the TaqI site that corresponds to amino acid #188, frame #3 (see FIG. 6; SEQ ID NO:6; and, SEQ ID NO:7). This created a unique BamHI site at that position. Because the precise position of the TaqI site could be assessed from the DNA sequence information available for the rsaA gene, the necessary translation frame was known and thus a single construction of a metallothionein gene was made. This was done by excision of the coding sequence of monkey metallothionein II peptide (60 amino acids comprising 10 cysteine residues and having a molecular weight of about 5000) at known restriction sites and adapting the gene ends with BamHI linkers with appropriate base pair spacers for the needed translation frame.

[0062] After insertion into the BamHI site created at position 188, frame 3, several clones were examined by determining whether they could bind elevated le...

example 3

Investigation of Other Permissive Sites in rsaA Gene

[0064] A library of 240 BamHI linker insertions was created using the procedures of Example 1. Of the 240 insertions, 45 target sites in the rsaA gene were made with TaqI. 34 of the latter insertions were discarded because the clones contained deletions of rsaA DNA as well as the linker insertions. The remaining 11 resulted in 5 non-permissive and the 6 permissive sites described in Example 1. The remaining 195 insertions in the library were made using the enzymes HinPI, AciI, and MspI to create target sites as outlined in Example 1. Of the latter 195 insertions, 49 permissive sites were located for a total of 55. Of those sites scored as non-permissive, some may have had deletions of rsaA DNA at the linker insertion site. One BamHI linker insertion at a TaqI site thought to be permissive was later found by nucleotide sequencing to be located outside the rsaA structural gene reducing the total number of permissive sites to 54 from ...

example 4

Further Studies with Cadmium Binding Polypeptides

[0066] The results described for Example 3 indicated that it would be possible to insert metallothionein at multiple places in the rsaA protein and thereby enhance the metal binding capacity of such a transformed Caulobacter. However, when the procedures of Example 2 were repeated to insert the metallothionein coding sequence into others of the 54 permissive sites identified in the preceding Example in each case, the transformed Caulobacter did not secrete a chimeric protein and did not synthesize an S-layer. Furthermore, the transformed Caulobacter of Example 2 was stable as long as the transformants were frozen immediately after isolation. When continuously cultured for approximately one week, the transformants deleted the metallothionein portion of the S-layer and the S-layer protein returns to its normal size.

[0067] Consideration of the predicted amino acid sequence of the rsaA protein shows that the latter protein lacks cysteine ...

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Abstract

DNA constructs are provided which code for at least the extreme C-terminal amino acids of the <highlight><uline>rsa< / uline>< / highlight>A protein of Caulobacter crescentus fused with heterologous polypeptides. Baterial cells containing, or which express the DNA constructs and secrete the resulting protein are also provided. Chimeric proteins including the C-terminal amino acids of the <highlight><uline>rsa< / uline>< / highlight>A protein are provided, including chimeric proteins comprising antigenic epitopes of the Infectious Hematopoietic Necrosis Virus.

Description

[0001] This application is a continuation-in-part of application Ser. No. 08 / 194,290 filed Feb. 9, 1994, which is a continuation-in-part of application Ser. No. 07 / 895,367 filed Jun. 9, 1992 now abandoned.[0002] This invention relates to the expression and secretion of heterologous peptides, from Caulobacter wherein the heterologous polypeptide is fused with the surface layer protein (S-layer protein) of the bacterium, or a portion of the S-layer protein.[0003] Bacterial surface proteins have been used as carriers for foreign (heterologous) polypeptides (particularly in Salmonella and E. coli) for various purposes, including the development of live vaccines. In some instances, the heterologous material is expressed as a fusion product with a surface protein of the bacterium. Generally, the use of such surface proteins as a vehicle for expression and / or presentation of heterologous polypeptides has been limited by the characteristics of a particular surface protein. The lipopolysacch...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00C07K14/145C07K14/21C12N1/21C12N15/31C12N15/74
CPCA61K39/00C07K14/005C07K14/21C07K2319/00C12N15/74C12N2760/20022
Inventor SMIT, JOHNBINGLE, WADE H.NOMELLINI, JOHN F.
Owner RES CORP TECH INC
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