Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)38105-5 ◽

1991 ◽

Vol 266 (10) ◽

pp. 6209-6214

Author(s):

M D Adams ◽

D J Maguire ◽

D L Oxender

Keyword(s):

Escherichia Coli ◽

Binding Proteins ◽

Binding Activity

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Biotin binding to avidin. Oligosaccharide side chain not required for ligand association

Biochemical Journal ◽

10.1042/bj2480167 ◽

1987 ◽

Vol 248 (1) ◽

pp. 167-171 ◽

Cited By ~ 117

Author(s):

Y Hiller ◽

J M Gershoni ◽

E A Bayer ◽

M Wilchek

Keyword(s):

Concanavalin A ◽

Sugar Content ◽

Binding Activity ◽

Side Chain ◽

Affinity Column ◽

Binding Properties ◽

Commercial Preparation ◽

Oligosaccharide Moiety ◽

Biotin Binding

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.

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Mannose-Binding Activity of Escherichia coli: a Determinant of Attachment and Ingestion of the Bacteria by Macrophages

Infection and Immunity ◽

10.1128/iai.29.2.417-424.1980 ◽

1980 ◽

Vol 29 (2) ◽

pp. 417-424

Author(s):

Zvi Bar-Shavit ◽

Rachel Goldman ◽

Itzhak Ofek ◽

Nathan Sharon ◽

David Mirelman

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Pathogenic Bacteria ◽

Binding Activity ◽

Exponential Phase ◽

Restrictive Temperature ◽

Filamentous Bacteria ◽

Phagocytic Cells ◽

E Coli ◽

Mannose Binding

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

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Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

Protein Engineering Design and Selection ◽

10.1093/protein/3.7.641 ◽

1990 ◽

Vol 3 (7) ◽

pp. 641-647 ◽

Cited By ~ 31

Author(s):

Helen Field ◽

G.T. Yarranton ◽

A.R. Rees

Keyword(s):

Escherichia Coli ◽

Heavy Chain ◽

Binding Activity ◽

Antigen Binding ◽

Variable Regions ◽

Mouse Immunoglobulin

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Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

Journal of Immunological Methods ◽

10.1016/j.jim.2014.03.010 ◽

2014 ◽

Vol 406 ◽

pp. 143-147 ◽

Cited By ~ 5

Author(s):

Raju Ahmed ◽

Emma Spikings ◽

Shaobo Zhou ◽

Andrew Thompsett ◽

Tiantian Zhang

Keyword(s):

Detection System ◽

Binding Activity ◽

Biotin Binding ◽

Endogenous Biotin

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The melibiose/Na + symporter of Escherichia coli : kinetic and molecular properties

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1990.0021 ◽

1990 ◽

Vol 326 (1236) ◽

pp. 411-423 ◽

Cited By ~ 44

Keyword(s):

Escherichia Coli ◽

Complex Cation ◽

Expression System ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Coupling Mechanism ◽

Facilitated Diffusion ◽

Histidine Residues ◽

Sugar Binding ◽

Dependent Transport

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H + , Na + or Li + -sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar—carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

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Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1607-1613.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1607-1613

Author(s):

M Matsuda ◽

B J Mayer ◽

H Hanafusa

Keyword(s):

Escherichia Coli ◽

Kinase Activity ◽

Binding Activity ◽

Transformed Cells ◽

Dependent Manner ◽

Tyrosine Kinase Activity ◽

Sh3 Domains ◽

Sh2 Domains ◽

Sarcoma Virus ◽

Avian Sarcoma

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.

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Novel application of streptavidin-hapten derivatives as protein-tracer conjugate in competitive-type immunoassays involving biotinylated detection probes

Clinical Chemistry ◽

10.1093/clinchem/37.1.58 ◽

1991 ◽

Vol 37 (1) ◽

pp. 58-63 ◽

Cited By ~ 8

Author(s):

M J Khosravi ◽

R C Morton

Keyword(s):

No Reduction ◽

Solid Phase ◽

Binding Activity ◽

Fluorescence Signal ◽

Antibody Immobilization ◽

Aggregate Formation ◽

Cross Link ◽

Time Resolved ◽

Biotin Binding ◽

Detection Reagent

Abstract To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.

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Mutants of Type II Heat-Labile Enterotoxin LT-IIa with Altered Ganglioside-Binding Activities and Diminished Toxicity Are Potent Mucosal Adjuvants

Infection and Immunity ◽

10.1128/iai.01009-06 ◽

2006 ◽

Vol 75 (2) ◽

pp. 621-633 ◽

Cited By ~ 20

Author(s):

Hesham F. Nawar ◽

Sergio Arce ◽

Michael W. Russell ◽

Terry D. Connell

Keyword(s):

Escherichia Coli ◽

Immune Responses ◽

Binding Activity ◽

Enzyme Linked Immunosorbent Assay ◽

Type I ◽

Type Ii ◽

Heat Labile ◽

Enhanced Expression ◽

Adhesin Protein ◽

And Function

ABSTRACT The structure and function LT-IIa, a type II heat-labile enterotoxin of Escherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.

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