fimh antibody cusabio

FimH-based display of functional eukaryotic proteins

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

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 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 membrane11,12. 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 membrane14,15.
  • 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 fibrillum17,18. 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-10ug

QP7372-ye-10ug 10ug
EUR 362

Recombinant E.coli Protein FimH Protein, His, Yeast-1mg

QP7372-ye-1mg 1mg
EUR 2747

Recombinant E.coli Protein FimH Protein, His, Yeast-200ug

QP7372-ye-200ug 200ug
EUR 1260

Recombinant E.coli Protein FimH Protein, His, Yeast-500ug

QP7372-ye-500ug 500ug
EUR 1804

Recombinant E.coli Protein FimH Protein, His, Yeast-50ug

QP7372-ye-50ug 50ug
EUR 480

Recombinant Escherichia coli FimH Protein (aa 22-300)

VAng-Lsx02377-1mgEcoli 1 mg (E. coli)
EUR 4793
Description: Escherichia coli protein FimH, recombinant protein.

Recombinant Escherichia coli FimH Protein (aa 22-300)

VAng-Lsx02377-500gEcoli 500 µg (E. coli)
EUR 3048
Description: Escherichia coli protein FimH, recombinant protein.

Recombinant Escherichia coli FimH Protein (aa 22-300)

VAng-Lsx02377-50gEcoli 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 100ug
EUR 707

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-10ug

QP7372-ec-10ug 10ug
EUR 326

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-1mg

QP7372-ec-1mg 1mg
EUR 2303

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-200ug

QP7372-ec-200ug 200ug
EUR 1115

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-500ug

QP7372-ec-500ug 500ug
EUR 1514

Recombinant E.coli Protein FimH Protein, His-GST, E.coli-50ug

QP7372-ec-50ug 50ug
EUR 435

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)

1-CSB-EP362349ENV
  • 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
  • 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
  • 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

Escherichia coli Type 1 fimbrin D-mannose specific adhesin (fimH)

1-CSB-YP362349ENV
  • 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
  • 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

H2B Antibody Antibody

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

CD11b Antibody Antibody

ABD2911 100 ug
EUR 438

anti- Antibody^Polyclonal antibody control antibody

LSMab09882 100 ug
EUR 438

Ly1 Antibody Reactive (LYAR) Antibody

20-abx008109
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Anti-Glycolipid Antibody (AGA) Antibody

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Ly1 Antibody Reactive (LYAR) Antibody

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Anti-Glycolipid Antibody (AGA) Antibody

abx036399-100ug 100 ug
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Ly1 Antibody Reactive (LYAR) Antibody

20-abx014333
  • EUR 314.00
  • EUR 98.00
  • EUR 398.00
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  • 100 ug
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Ly1 Antibody Reactive (LYAR) Antibody

abx033330-400ul 400 ul
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Ly1 Antibody Reactive (LYAR) Antibody

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Anti-Glycoprotein Antibody (GP) Antibody

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Anti-Glycoprotein Antibody (GP) Antibody

20-abx319901
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Anti-Glycoprotein Antibody (GP) Antibody

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Anti-Glycoprotein Antibody (GP) Antibody

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Ly1 Antibody Reactive (LYAR) Antibody

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Ly1 Antibody Reactive (LYAR) Antibody

20-abx324434
  • EUR 314.00
  • EUR 244.00
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Ly1 Antibody Reactive (LYAR) Antibody

20-abx311665
  • EUR 411.00
  • EUR 1845.00
  • EUR 599.00
  • EUR 182.00
  • EUR 300.00
  • 100 ug
  • 1 mg
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  • 50 ug
  • Shipped within 5-10 working days.