Details

Project TitleCAULOBACTER S-LAYER KITS: For Recombinant Protein Secretion AND Peptide/Protein Display
Track Code92-039
Short Description

A novel protein secretion and display system, using Caulobacter crescentus, that provides scalable, inexpensive secretion of recombinant proteins (via fusion with a C-terminal secretion signal) or display of peptides or proteins on the cell surface (via insertion into the complete S-layer gene sequence). Caulobacter expressing the immunoglobulin G-binding domain of Streptococcal Protein G is available for purchase.

AbstractNone
 
Tagsbiomedical, biotechnology, life sciences, medical sciences, microbial biotechnology, r&d discovery, research tools, science & technology, vaccine development, vaccines
 
Posted DateApr 12, 2010 2:21 PM

Description

Caulobacter expressing the immunoglobulin G-binding domain of Streptococcal Protein G is available for purchase.

 

• A portion of the streptooccal protein G coding sequence, including the IgG-binding domain GB1, was inserted into RsaA. The Caulobacter displays 3 copies of the GB1 sequence in tandem.

• Recombinant protein G-Caulobacter cells function as immunoreactive reagents. Their performance is comparable to that of protein G-Sepharose beads.

•No plasmids, no drug selection, and growth in standard media.

 

The Caulobacter S-layer Expression system is available as two separate kits

BENEFITS OF THE CAULOBACTER S-LAYER PROTEIN SECRETION KIT:
• Production is easily scalable using simple media/fermentation systems and inexpensive batch culture that contains no animal or plant proteins.
Caulobacter secreted proteins can be retrieved by conventional methods to concentrate protein from the medium, especially as there are no proteases and very low levels of other cellular proteins. Alternatively, a slow shaking method results in an aggregation of caulobacter S-layer fusion proteins and is readily collected by simple filtration with up to 95% purity.
• Large quantities (10% of total cell protein) of a wide range of secreted proteins can be produced; a typical yield is 100mg/ml for low density batch cultures. however, yields of 500-700mg/ml can be obtained.
• Proteins ranging from 2-70 KDa have been successfully expressed, without post-translational glycosylation.
• Export system is capable of tolerating a wide variety of foreign proteins and is stable in expressing repeated polymer sequences.
• The endotoxin potential of the Caulobacter LPS is approximately 100-1000 times lower than E. coli.
• Cloning vehicle is a single high copy number vector that replicates in Caulobacter as well as E. coli cloning strains.

BENEFITS OF THE CAULOBACTER S-LAYER PROTEIN DISPLAY KIT:
• Display of peptides from 10-300 amino acids in length has been accomplished with a high rate of success.
• Multiple peptides/proteins can be displayed on the same bacterium.
• The genetics and biochemistry of Caulobacter is well established, allowing easy engineering of the system.
• Display of discrete or random peptide or gene segment libraries is possible.
Caulobacters are large enough to be detected and utilized in a Fluorescence Activated Cell Sorter.
Caulobacter can be injected into animals without evidence of sepsis and may be a useful platform for animal and fish vaccine development by display of antigens and immunomodulators.

Potential Applications

The Cauolobacter fusion secretion kit can be used for the large scale production of recombinant fusion proteins where the target protein can be fused to the C-terminal secretion signal of the S-layer gene and subsequently secreted into the surrounding medium, allowing for simple retrieval. Examples of successfully secreted proteins include large portions of Anthrax Protective antigen (144/239 aa) and bacterial cellulase (369 aa).

 

The Caulobacter protein display kit is capable of displaying peptides of around 150 amino acids and there are numerous examples of still larger sizes. Approximately 40-50 different peptides have been presented to date, including vaccine epitopes, immunodiagnostic particles and anti-infective agents (blocking peptides). Examples of displayed peptides include an envelope protein from Infectious Hematopoietic Necrosis virus (184 aa), Protein G IgG binding domains (275 aa) and human and mouse GM CSF (~125 aa), and several single chain antibodies (~275 aa).

State of Development

This technology is fully developed and validated. The technology has been cited in many publications, including the following :

•Nomellini et al 2007. S-layer-mediated display of the immunoglobulin G-binding domain of streptococcal protein G on the surface of caulobacter crescentus: development of an immunoactive reagent. Appl Environ Microbiol. May;73(10):3245-53

•Bingle et al. 2000. Secretion of the Caulobacter crescentus S-layer Protein: Further localization of the C-terminal secretion signal and its use for secretion of recombinant proteins. J. Bacteriol. 182: 3298-3301.

•Duncan et al, 2005. Evaluating a new system for the development of particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus; fusion with the ß-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi yields a robust, catalytically active product. Appl. Biochem. Biotechnol. 127: 95-110.

Additional

Caulobacter expressing streptococcal protein G on the surface is available for purchase

Both Caulobacter Protein Secretion and Peptide Display systems are commercially available as kits.

To review the license or purchase a product, please click "license" below and follow the prompts.

NOTE: Miscellaneous and individual components of these kits are also available commercially-Please download the MTA/PURCHASE REQUEST FORM from the "download" section and proceed as stated in the form.

Background

The protein expression system uses Caulobacter crescentus, a harmless gram-negative, non-pathogenic, aerobic bacteria. The Caulobacter produces and secretes a single expressed protein, RsaA, that assembles on its outer surface, forming a two-dimensional paracrystalline array or S-layer.

 

Files

File Name Description
**MTA/PURCHASE ORDER FORM**-Miscellaneous kit Components ONLY None Download
p4A 723_ c-Myc seq.pdf None Download
p336C seq.doc None Download
Protein and Peptide Display Kit.pdf None Download
Fusion Protein Secretion Kit.pdf None Download
Caulobacter Protein and Peptide Display Manual.pdf None Download
Caulobacter Fusion Protein Secretion Manual.pdf None Download

Offerings

Name Price
Caulobacter Expressing Streptococcal Protein G - 1 stab 125.00 USD Buy
Caulobacter S-Layer Kit: Fusion Protein Secretion System 450.00 USD Buy
Caulobacter S-Layer Kit: Protein/Peptide Display System 350.00 USD Buy
Caulobacter strain-B5BAC cells 200.00 USD Buy
Caulobacter strain-deltaP6BAC cells 200.00 USD Buy

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