FimH adhesin to display the preS2 domain of the hepatitis B

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.

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.

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.

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 biosensors², development of novel vaccine platforms³, screening of antibody libraries⁴ and whole-cell biocatalysis⁵.

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 bacteria⁶. 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 activities⁷. A similar experience was made when displaying sorbitol dehydrogenase⁸.
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-100ug
QP7372-ye-100ug	EnQuireBio	100ug	EUR 945.6

Recombinant E.coli Protein FimH Protein, His, Yeast-200ug
QP7372-ye-200ug	EnQuireBio	200ug	EUR 1512

Recombinant E.coli Protein FimH Protein, His, Yeast-500ug
QP7372-ye-500ug	EnQuireBio	500ug	EUR 2164.8

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-1mg
QP7372-ec-1mg	EnQuireBio	1mg	EUR 2763.6

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-10ug
QP7372-ec-10ug	EnQuireBio	10ug	EUR 391.2

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-50ug
QP7372-ec-50ug	EnQuireBio	50ug	EUR 522

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-100ug
QP7372-ec-100ug	EnQuireBio	100ug	EUR 848.4

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-200ug
QP7372-ec-200ug	EnQuireBio	200ug	EUR 1338

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-500ug
QP7372-ec-500ug	EnQuireBio	500ug	EUR 1816.8

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)
1-CSB-EP362349ENV	Cusabio	EUR 733.20 EUR 370.80 EUR 2192.40 EUR 1126.80 EUR 1461.60 EUR 476.40	100ug 10ug 1MG 200ug 500ug 50ug

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 733.20 EUR 370.80 EUR 2192.40 EUR 1126.80 EUR 1461.60 EUR 476.40	100ug 10ug 1MG 200ug 500ug 50ug

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 733.20 EUR 370.80 EUR 2192.40 EUR 1126.80 EUR 1461.60 EUR 476.40	100ug 10ug 1MG 200ug 500ug 50ug

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-YP362349ENV	Cusabio	EUR 814.80 EUR 402.00 EUR 2606.40 EUR 1261.20 EUR 1730.40 EUR 522.00	100ug 10ug 1MG 200ug 500ug 50ug

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 954.00 EUR 541.20 EUR 2745.60 EUR 1400.40 EUR 1869.60 EUR 661.20	100ug 10ug 1MG 200ug 500ug 50ug

Description: Recombinant Escherichia coli Type 1 fimbrin D-mannose specific adhesin(fimH) expressed in Yeast

fim3 Antibody
1-CSB-PA322971LA01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim3. Recognizes fim3 from Bordetella pertussis. This antibody is Unconjugated. Tested in the following application: ELISA

fim2 Antibody
1-CSB-PA356626LA01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim2. Recognizes fim2 from Bordetella pertussis. This antibody is Unconjugated. Tested in the following application: ELISA

fim2 Polyclonal Antibody
A54740	EpiGentek	EUR 684.66 EUR 117.70 EUR 302.50 EUR 423.50	100 µg 20 ul 50 ul 100 ul

fim3 Antibody, HRP conjugated
1-CSB-PA322971LB01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim3. Recognizes fim3 from Bordetella pertussis. This antibody is HRP conjugated. Tested in the following application: ELISA

fim2 Antibody, HRP conjugated
1-CSB-PA356626LB01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim2. Recognizes fim2 from Bordetella pertussis. This antibody is HRP conjugated. Tested in the following application: ELISA

fim3 Antibody, FITC conjugated
1-CSB-PA322971LC01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim3. Recognizes fim3 from Bordetella pertussis. This antibody is FITC conjugated. Tested in the following application: ELISA

fim2 Antibody, FITC conjugated
1-CSB-PA356626LC01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim2. Recognizes fim2 from Bordetella pertussis. This antibody is FITC conjugated. Tested in the following application: ELISA

fim3 Antibody, Biotin conjugated
1-CSB-PA322971LD01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim3. Recognizes fim3 from Bordetella pertussis. This antibody is Biotin conjugated. Tested in the following application: ELISA

fim2 Antibody, Biotin conjugated
1-CSB-PA356626LD01BUA	Cusabio	EUR 380.40 EUR 402.00	100ug 50ug

Description: A polyclonal antibody against fim2. Recognizes fim2 from Bordetella pertussis. This antibody is Biotin conjugated. Tested in the following application: ELISA

fim2 Polyclonal Antibody, HRP Conjugated
A54739	EpiGentek	EUR 684.66 EUR 302.50 EUR 423.50	100 µg 50 ul 100 ul

fim2 Polyclonal Antibody, FITC Conjugated
A54738	EpiGentek	EUR 684.66 EUR 302.50 EUR 423.50	100 µg 50 ul 100 ul

fim2 Polyclonal Antibody, Biotin Conjugated
A54737	EpiGentek	EUR 684.66 EUR 302.50 EUR 423.50	100 µg 50 ul 100 ul

CS6 Fimbrial Subunit B (CSSB) Antibody
20-abx300674	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Serotype 2 fimbriasubunit Antibody
20-abx110520	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Fimbrial Subunit Type 3 (mrkA) Antibody
20-abx318912	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Serotype 3 fimbrial subunit Antibody
20-abx110386	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

CFA/I fimbrial subunit E (CFAE) Antibody
20-abx300666	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

CS6 Fimbrial Subunit B (CSSB) Antibody (HRP)
20-abx300675	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Serotype 2 fimbriasubunit Antibody (HRP)
20-abx109070	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Fimbrial Subunit Type 3 (MRKA) Antibody (HRP)
20-abx318913	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

CS6 Fimbrial Subunit B (CSSB) Antibody (FITC)
20-abx300676	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

CFA/I fimbrial subunit E (CFAE) Antibody (HRP)
20-abx300667	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Serotype 3 fimbrial subunit Antibody (HRP)
20-abx108940	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug

Serotype 2 fimbriasubunit Antibody (FITC)
20-abx107653	Abbexa	EUR 493.20 EUR 2214.00 EUR 718.80 EUR 218.40 EUR 360.00	100 ug 1 mg 200 ug 20 ug 50 ug