Purification of Filamentous Bacteriophage for Phage Display using Size-Exclusion Chromatography

Захарова, М. В.; Kozyr, A. V.; Ignatova, A. N.; Vinnikov, Ilya A.; Shemyakin, I. G.; Kolesnikov, Alexander V.

doi:10.2144/05382bm04

Cited by 27 publications

(18 citation statements)

References 8 publications

(10 reference statements)

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“…Ion exchange chromatofocusing is possible, but would requires determination of the phage pI and stability of the phage through a pH range for each phage system under study 25 . Sephacryl S-500 size exclusion chromatography (SEC) has also been demonstrated, 26 but most phages would be larger than its exclusion limit and would elute in the void volume with other large contaminants.…”

Section: Introductionmentioning

confidence: 99%

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria

et al. 2012

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Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/μm2 capturing 18.0 ± 0.3 bacteria/100 μm2. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface.

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Section: Introductionmentioning

confidence: 99%

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria

et al. 2012

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“…Zakharova and co-workers (3) have recently shown that size-exclusion chromatography (SEC) in Sephacryl S-500 resin (nominal size-exclusion limit: 20 MDa) can serve as an effective alternative to CsCl equilibrium density gradient centrifugation for purification of PEG-precipitated filamentous virions. Virions emerge in the void volume while protein contaminants emerge as a broad included peak.…”

Section: Introductionmentioning

confidence: 99%

Hydroxyapatite Chromatography of Phage-Display Virions

Smith

Gingrich

2005

BioTechniques

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Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.

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“…These values were utilized in future ELISAs to calculate the molar concentrations employed. We selected 1.7 × 10 5 kDa as the approximate molecular weight of the phage particle …”

Section: Methodsmentioning

confidence: 99%

A Sensitive phage‐based capture ELISA for sub‐femtomolar detection of protein variants directly from biological samples

Williams

Schulz

Sierks

2014

Biotechnology Progress

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To determine the role of proteins, and in particular protein variants, in human health, it may often be necessary to quantitatively determine the concentration of a specific protein variant present in complex biological samples such as blood, cerebral spinal fluid (CSF), or tissue. Many protein variants are present only at trace levels and therefore a simple assay with very high sensitivity and reliability would greatly facilitate correlation of the presence of particular protein variants with the progression of specific diseases. We have developed a simple phage based capture ELISA system that enables femtomolar or better detection of individual protein variants directly from complex biological samples. The protocol utilizes a capture reagent that selectively recognizes a unique epitope of the protein variant and a phage based detection reagent that binds to a second epitope present in all forms of the target protein. The phage based detection reagent is essentially a self-assembling nanoparticle consisting of several thousand coat proteins that can each be labeled to amplify the detection signal by several orders of magnitude. Here we demonstrate that we can achieve subfemtomolar detection of individual protein variants that have been implicated in neurodegenerative disease directly from complex tissue homogenates and sera. The ELISA system should facilitate identification of disease specific protein variants or other compounds even when present at trace amounts in samples including blood, CSF, saliva and urine.

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Purification of Filamentous Bacteriophage for Phage Display using Size-Exclusion Chromatography

Cited by 27 publications

References 8 publications

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria

Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria

Hydroxyapatite Chromatography of Phage-Display Virions

A Sensitive phage‐based capture ELISA for sub‐femtomolar detection of protein variants directly from biological samples

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