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

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 Escherichia coli FimH Protein (aa 22-300)
VAng-Lsx02377-1mgEcoli	Creative Biolabs	1 mg (E. coli)	EUR 5751.6
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 3657.6
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 1083.6
Description: Escherichia coli protein FimH, recombinant protein.

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-10ug
QP7372-ec-10ug	EnQuireBio	10ug	EUR 391.2

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-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

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

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

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

Positive control tissue section for each individua
Control-Slides-for-each-antibody	Innovex	Set of 25	EUR 355
Description: Positive control tissue section for each individual antibody; Based on availability; INQUIRE

ASAP1 antibody Antibody
DF8746	Affbiotech	200ul	EUR 420

CD11b Antibody Antibody
ABD2911	Lifescience Market	100 ug	EUR 525.6

anti- Antibody^Polyclonal antibody control antibody
LSMab09882	Lifescience Market	100 ug	EUR 525.6

ARHGDIA Antibody / RHOGDI Antibody
F54788-0.08ML	NSJ Bioreagents	0.08 ml	EUR 165

ARHGDIA Antibody / RHOGDI Antibody
F54788-0.4ML	NSJ Bioreagents	0.4 ml	EUR 379

Antibody
A1360-500	Biovision	each	Ask for price

Ly1 Antibody Reactive (LYAR) Antibody
20-abx123734	Abbexa	EUR 493.20 EUR 710.40	100 ul 200 ul

Anti-Glycolipid Antibody (AGA) Antibody
20-abx004855	Abbexa	EUR 493.20 EUR 710.40 EUR 218.40 EUR 376.80	100 ul 200 ul 20 ul 50 ul

Ly1 Antibody Reactive (LYAR) Antibody
20-abx014333	Abbexa	EUR 376.80 EUR 117.60 EUR 477.60 EUR 594.00	100 ug 10 ug 200 ug 300 µg

Ly1 Antibody Reactive (LYAR) Antibody
20-abx008109	Abbexa	EUR 360.00 EUR 526.80 EUR 226.80	100 ul 200 ul 30 ul

Ly1 Antibody Reactive (LYAR) Antibody
abx033330-400ul	Abbexa	400 ul	EUR 627.6

Ly1 Antibody Reactive (LYAR) Antibody
abx033330-80l	Abbexa	80 µl	EUR 343.2

Anti-Glycolipid Antibody (AGA) Antibody
abx036399-100ug	Abbexa	100 ug	EUR 469.2

Anti-Glycolipid Antibody (AGA) Antibody
abx230204-100ug	Abbexa	100 ug	EUR 577.2

Anti-Glycoprotein Antibody (GP) Antibody
20-abx319900	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

Anti-Glycoprotein Antibody (GP) Antibody
20-abx319901	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

Anti-Glycoprotein Antibody (GP) Antibody
20-abx319905	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

Anti-Glycoprotein Antibody (GP) Antibody
20-abx319913	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

Ly1 Antibody Reactive (LYAR) Antibody
20-abx311665	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

Ly1 Antibody Reactive (LYAR) Antibody
20-abx324434	Abbexa	EUR 376.80 EUR 292.80	100 ug 50 ug

Ly1 Antibody Reactive (LYAR) Antibody
abx234901-100ug	Abbexa	100 ug	EUR 661.2

Anti-Anti-SEPT6 antibody antibody
STJ11100949	St John's Laboratory	100 µl	EUR 332.4
Description: This gene is a member of the septin family of GTPases. Members of this family are required for cytokinesis. One version of pediatric acute myeloid leukemia is the result of a reciprocal translocation between chromosomes 11 and X, with the breakpoint associated with the genes encoding the mixed-lineage leukemia and septin 2 proteins. This gene encodes four transcript variants encoding three distinct isoforms. An additional transcript variant has been identified, but its biological validity has not been determined.

Anti-Anti-SEPT9 Antibody antibody
STJ111369	St John's Laboratory	100 µl	EUR 332.4
Description: This gene is a member of the septin family involved in cytokinesis and cell cycle control. This gene is a candidate for the ovarian tumor suppressor gene. Mutations in this gene cause hereditary neuralgic amyotrophy, also known as neuritis with brachial predilection. A chromosomal translocation involving this gene on chromosome 17 and the MLL gene on chromosome 11 results in acute myelomonocytic leukemia. Multiple alternatively spliced transcript variants encoding different isoforms have been described.