FimH eukaryotic proteins on bacteria surfaces - Heraeus

Abstract

The demand for recombinant proteins for analytic and therapeutic purposes is increasing; however, most currently used bacterial production systems accumulate the recombinant proteins in the intracellular space, which requires denaturating procedures for harvesting and functional testing. We here present a novel FimH-based expression system that enables display of fully functional eukaryotic proteins while preventing technical difficulties in translocating, folding, stabilizing and isolating the displayed proteins. As examples, Gaussia Luciferase (GLuc), epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and epiregulin (EPRG) were expressed as FimH fusion proteins on the surface of E. coli bacteria.

The fusion proteins were functionally active and could be released from the bacterial surface by specific proteolytic cleavage into the culture supernatant allowing harvesting of the produced proteins. EGFR ligands, produced as FimH fusion proteins and released by proteolytic cleavage, bound to the EGF receptor (EGFR) on cancer cells inducing EGFR phosphorylation.

In another application of the technology, GLuc-FimH expressed on the surface of bacteria was used to track tumor-infiltrating bacteria by bioluminescence imaging upon application to mice, thereby visualizing the colonization of transplanted tumors. The examples indicate that the FimH-fusion protein technology can be used in various applications that require functionally active proteins to be displayed on bacterial surfaces or released into the culture supernatant.

fimh antibody cusabio

Introduction

Bacterial surface display of recombinant proteins has become an attractive strategy for a broad range of applications such as production of bioadsorbents¹, generation of cellular biosensors, development of novel vaccine platforms³, screening of antibody libraries⁴ and whole-cell biocatalysis.

Generally, the procedure requires the fusion of the protein-of-interest (POI) to a bacterial surface protein to display the POI on the surface of the genetically modified bacteria. Several surface-anchoring motifs like LPP-OmpA, LamB, PhoE, ice nucleation protein (INP) and auto-transporter are employed as carrier proteins for crossing the bacteria membrane.

Despite the successful approaches, several problems remain to be solved, including the substantially reduced functional activity of the displayed proteins. Compared with their soluble form, surface-anchored β-lactamase fused to the translation unit (TU) of an auto-transporter shows substantially reduced catalytic activities. A similar experience was made when displaying sorbitol dehydrogenase.
A major problem in the use of auto-transporters arises from the tertiary structure of the passenger domains and the size of the central cavity that permits translocating only small proteins. It seems not only to be a matter of size since even the 62 amino acids protein aprotinin is not efficiently translocated through the outer membrane⁹.
Translocation by auto-transporters is very sensitive to structure of the passenger proteins that consist of a β-strands backbone with at least 300 amino acids thereby substantially limiting the applicability to variety of potential cargos¹⁰.
As an alternative approach, protein sequences derived from the major E. coli lipoprotein (Lpp) were fused to the N-terminus of the POI to direct the protein to the outer membrane^11,12. The system consists of two key anchoring motifs; the Lpp-derived signal sequence at the N-terminus to target the fusion protein to the inner surface of the outer membrane, and the outer membrane protein A (OmpA)-derived transmembrane region to transfer the protein across the outer membrane¹².
Since its introduction by Ghrayeb and Inouye¹³ in 1984, the Lpp-OmpA display method is facing difficulties including the low expression rate and the insufficient translocation efficiency which may be due to steric hindrance and incorrect folding when anchoring in the outer membrane^14,15.
In Gram-negative bacteria the outer membrane generally acts as a barrier to restrict the protein export from the cell interior; only pilins, flagellins, specific surface enzymes, and a few bacterial toxins are transported across the outer membrane¹⁶.
These natural display systems have the benefit of being optimized for transporting and folding protein units to build polymeric structures on the extracellular surface making the display system attractive for biotechnological applications. We here used the fimbriae protein FimH, the mannose-specific adhesin of the E. coli type-1 fimbriae, for the extracellular display of recombinant proteins.
Type-1 fimbriae are composed of up to 3,000 copies of the subunit FimA, that form the pilus rod, as well as the subunits FimF, FimG and FimH building the distal tip fibrillum^17,18. In initial studies, Pallesen and colleagues used the positions 225 and 258 within the FimH adhesin to display the preS2 domain of the hepatitis B surface antigen or an epitope from cholera toxin¹⁹.
Both positions within the FimH protein proved to be suitable for the integration of peptides of up to 56 amino acids which could be produced, displayed on the cell surface and partially conserved the adhesive function of FimH¹⁹.
Longer peptide or full length proteins displayed by FimH in that position were so far not reported. While short polypeptides used for vaccines could be displayed, the technique failed in functionally expressing large proteins like enzymes or cytokines.
Here we identified alternative positions within the FimH protein to display larger proteins in a functionally active fashion. Based on the 3D modelling of E. coli type-1 pili²⁰ we identified the N-terminus of the FimH domain on the fimbriae tip as a suitable integration site of a larger protein.
As examples, we genetically linked Gaussia luciferase (GLuc) and human epidermal growth factor (EGF), tumor growth factor-a (TGF-α) and epiregulin (EREG), all ligands of the epidermal growth factor receptor (EGFR), to FimH.
Expressed by transformed E. coli, the proteins conserved their functional capacities. In particular, GLuc-FimH displaying E. coli bacteria were tracked during colonization of syngeneic tumors in an immunocompetent mouse model of pancreatic cancer during a six week period without losing GLuc activity.
Bacteria with surface displayed proteins can be used for screening purposes and, furthermore, can be released in a functionally active form by specific proteolytic cleavage making the strategy attractive for protein production without the need to disrupt the bacteria by harsh procedures.

Recombinant E.coli Protein FimH Protein, His, Yeast-1mg
QP7372-ye-1mg	EnQuireBio	1mg	EUR 2747

Recombinant E.coli Protein FimH Protein, His, Yeast-200ug
QP7372-ye-200ug	EnQuireBio	200ug	EUR 1260

Recombinant E.coli Protein FimH Protein, His, Yeast-500ug
QP7372-ye-500ug	EnQuireBio	500ug	EUR 1804

Recombinant E.coli Protein FimH Protein, His, Yeast-50ug
QP7372-ye-50ug	EnQuireBio	50ug	EUR 480

Recombinant Escherichia coli FimH Protein (aa 22-300)
VAng-Lsx02377-1mgEcoli	Creative Biolabs	1 mg (E. coli)	EUR 4793
Description: Escherichia coli protein FimH, recombinant protein.

Recombinant Escherichia coli FimH Protein (aa 22-300)
VAng-Lsx02377-500gEcoli	Creative Biolabs	500 µg (E. coli)	EUR 3048
Description: Escherichia coli protein FimH, recombinant protein.

Recombinant Escherichia coli FimH Protein (aa 22-300)
VAng-Lsx02377-50gEcoli	Creative Biolabs	50 µg (E. coli)	EUR 903
Description: Escherichia coli protein FimH, recombinant protein.

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-100ug
QP7372-ec-100ug	EnQuireBio	100ug	EUR 707

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-10ug
QP7372-ec-10ug	EnQuireBio	10ug	EUR 326

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-1mg
QP7372-ec-1mg	EnQuireBio	1mg	EUR 2303

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-200ug
QP7372-ec-200ug	EnQuireBio	200ug	EUR 1115

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-500ug
QP7372-ec-500ug	EnQuireBio	500ug	EUR 1514

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-50ug
QP7372-ec-50ug	EnQuireBio	50ug	EUR 435

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)
1-CSB-YP362349ENV	Cusabio	EUR 679.00 EUR 335.00 EUR 2172.00 EUR 1051.00 EUR 1442.00 EUR 435.00	100ug 10ug 1MG 200ug 500ug 50ug
MW: 31.1 kDa Buffer composition: Tris-based buffer with 50% glycerol.
Description: Recombinant Escherichia coli Type 1 fimbrin D-mannose specific adhesin(fimH) expressed in Yeast

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)
1-CSB-YP362349ENVe1	Cusabio	EUR 795.00 EUR 451.00 EUR 2288.00 EUR 1167.00 EUR 1558.00 EUR 551.00	100ug 10ug 1MG 200ug 500ug 50ug
MW: 29.1 kDa Buffer composition: Tris-based buffer with 50% glycerol.
Description: Recombinant Escherichia coli Type 1 fimbrin D-mannose specific adhesin(fimH) expressed in Yeast

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)
1-CSB-EP362349ENV	Cusabio	EUR 611.00 EUR 309.00 EUR 1827.00 EUR 939.00 EUR 1218.00 EUR 397.00	100ug 10ug 1MG 200ug 500ug 50ug
MW: 59.1 kDa Buffer composition: Tris-based buffer with 50% glycerol.
Description: Recombinant Escherichia coli Type 1 fimbrin D-mannose specific adhesin(fimH) expressed in E.coli

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)
1-CSB-EP362349ENVa0	Cusabio	EUR 611.00 EUR 309.00 EUR 1827.00 EUR 939.00 EUR 1218.00 EUR 397.00	100ug 10ug 1MG 200ug 500ug 50ug
MW: 33.1kDa Buffer composition: Tris-based buffer with 50% glycerol.
Description: Recombinant Escherichia coli Type 1 fimbrin D-mannose specific adhesin(fimH) expressed in E.coli

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)
1-CSB-EP362349ENVc7	Cusabio	EUR 611.00 EUR 309.00 EUR 1827.00 EUR 939.00 EUR 1218.00 EUR 397.00	100ug 10ug 1MG 200ug 500ug 50ug
MW: 29.8 kDa Buffer composition: Tris-based buffer with 50% glycerol.
Description: Recombinant Escherichia coli Type 1 fimbrin D-mannose specific adhesin(fimH) expressed in E.coli

H2B Antibody Antibody
AF4659	Affbiotech	200ul	EUR 376
Description: H2B Antibody Antibody detects endogenous levels of H2B.

CD11b Antibody Antibody
ABD2911	Lifescience Market	100 ug	EUR 438

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LSMab09882	Lifescience Market	100 ug	EUR 438

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20-abx123734	Abbexa	EUR 411.00 EUR 592.00	100 ul 200 ul
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Ly1 Antibody Reactive (LYAR) Antibody
20-abx311665	Abbexa	EUR 411.00 EUR 1845.00 EUR 599.00 EUR 182.00 EUR 300.00	100 ug 1 mg 200 ug 20 ug 50 ug
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abx234901-100ug	Abbexa	100 ug	EUR 551
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abx230204-100ug	Abbexa	100 ug	EUR 481
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20-abx324434	Abbexa	EUR 314.00 EUR 244.00	100 ug 50 ug
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20-abx004855	Abbexa	EUR 411.00 EUR 592.00 EUR 182.00 EUR 314.00	100 ul 200 ul 20 ul 50 ul
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abx036399-100ug	Abbexa	100 ug	EUR 391
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abx033330-400ul	Abbexa	400 ul	EUR 523
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Anti-Anti-SEPT2 Antibody antibody
STJ25475	St John's Laboratory	100 µl	EUR 277

Anti-Anti-SEPT5 Antibody antibody
STJ25477	St John's Laboratory	100 µl	EUR 277
Description: This gene is a member of the septin gene family of nucleotide binding proteins, originally described in yeast as cell division cycle regulatory proteins. Septins are highly conserved in yeast, Drosophila, and mouse and appear to regulate cytoskeletal organization. Disruption of septin function disturbs cytokinesis and results in large multinucleate or polyploid cells. This gene is mapped to 22q11, the region frequently deleted in DiGeorge and velocardiofacial syndromes. A translocation involving the MLL gene and this gene has also been reported in patients with acute myeloid leukemia. Alternative splicing results in multiple transcript variants. The presence of a non-consensus polyA signal (AACAAT) in this gene also results in read-through transcription into the downstream neighboring gene (GP1BB; platelet glycoprotein Ib), whereby larger, non-coding transcripts are produced.

Anti-Anti-SEPT8 Antibody antibody
STJ25479	St John's Laboratory	100 µl	EUR 277
Description: This gene is a member of the septin family of nucleotide binding proteins, originally described in yeast as cell division cycle regulatory proteins. Septins are highly conserved in yeast, Drosophila, and mouse, and appear to regulate cytoskeletal organization. Disruption of septin function disturbs cytokinesis and results in large multinucleate or polyploid cells. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene.

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STJ28365	St John's Laboratory	100 µl	EUR 277

Anti-Anti-SEPT7 Antibody antibody
STJ28963	St John's Laboratory	100 µl	EUR 277
Description: This gene encodes a protein that is highly similar to the CDC10 protein of Saccharomyces cerevisiae. The protein also shares similarity with Diff 6 of Drosophila and with H5 of mouse. Each of these similar proteins, including the yeast CDC10, contains a GTP-binding motif. The yeast CDC10 protein is a structural component of the 10 nm filament which lies inside the cytoplasmic membrane and is essential for cytokinesis. This human protein functions in gliomagenesis and in the suppression of glioma cell growth, and it is required for the association of centromere-associated protein E with the kinetochore. Alternative splicing results in multiple transcript variants. Several related pseudogenes have been identified on chromosomes 5, 7, 9, 10, 11, 14, 17 and 19.

Anti-Anti-SEPT8 Antibody antibody
STJ117206	St John's Laboratory	100 µl	EUR 277
Description: This gene is a member of the septin family of nucleotide binding proteins, originally described in yeast as cell division cycle regulatory proteins. Septins are highly conserved in yeast, Drosophila, and mouse, and appear to regulate cytoskeletal organization. Disruption of septin function disturbs cytokinesis and results in large multinucleate or polyploid cells. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene.

Anti-Anti-SEPT12 Antibody antibody
STJ117759	St John's Laboratory	100 µl	EUR 277
Description: This gene encodes a guanine-nucleotide binding protein and member of the septin family of cytoskeletal GTPases. Septins play important roles in cytokinesis, exocytosis, embryonic development, and membrane dynamics. Multiple transcript variants encoding different isoforms have been found for this gene.

Anti-Anti-MARCH6 Antibody antibody
STJ118549	St John's Laboratory	100 µl	EUR 277

Anti-Anti-MARCH6 Antibody antibody
STJ118550	St John's Laboratory	100 µl	EUR 277

Anti-Anti-MARCH7 Antibody antibody
STJ118752	St John's Laboratory	100 µl	EUR 277

Anti-Anti-SEPT3 Antibody antibody
STJ118990	St John's Laboratory	100 µl	EUR 277

Anti-Anti-SEPT1 antibody antibody
STJ119580	St John's Laboratory	100 µl	EUR 277
Description: This gene is a member of the septin family of GTPases. Members of this family are required for cytokinesis and the maintenance of cellular morphology. This gene encodes a protein that can form homo- and heterooligomeric filaments, and may contribute to the formation of neurofibrillary tangles in Alzheimer's disease. Alternatively spliced transcript variants have been found but the full-length nature of these variants has not been determined. [provided by RefSeq, Dec 2012]