A Method of Immobilization on the Solid Support of Complex and Simple Enzymes Retaining Their Activity

Chernukhin, Igor; Klenova, Elena

doi:10.1006/abio.2000.4521

Cited by 8 publications

(4 citation statements)

References 20 publications

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“…The resulting complexes therefore contain DNA fragments associated with the two partner proteins. The matrices containing the conjugated anti-CTCF, anti-LS Pol II antibody (N-20), or preimmune serum were prepared as previously described (9). The DNA purified from ChIP assays was measured, and 1 to 10 l of the DNA was used for PCR amplification.…”

Section: Methodsmentioning

confidence: 99%

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Chernukhin

Shamsuddin

Kang

et al. 2007

Molecular and Cellular Biology

154

130

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CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

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Section: Methodsmentioning

confidence: 99%

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Chernukhin

Shamsuddin

Kang

et al. 2007

Molecular and Cellular Biology

154

130

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“…Dimethyl pimelimidate (DMP) is a homobifunctional, crosslinking agent that may be used to couple proteins to aminated surfaces via reaction with primary amines on proteins, while introducing a 7‐carbon spacer. This water soluble molecule is readily available and holds potential for biomaterial applications, as shown by previous studies in which it was used for the effective immobilization of enzymes, while preserving enzyme activity and stability 17, 18…”

Section: Introductionmentioning

confidence: 99%

Enhanced smooth muscle cell adhesion and proliferation on protein‐modified polycaprolactone‐based copolymers

Bramfeldt

Vermette

2008

J Biomedical Materials Res

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Smooth muscle cells (SMC) were cultured for up to 6 days on copolymer films fabricated from a PCL-PEG-PCL block copolymer or P(epsilon-CL-co-D,L-LA)-PEG-P(epsilon-CL-co-D,L-LA), named P(100/0) and P(70/30), respectively. The films were modified by aminolysis using 1,6-hexanediamine, and fibronectin, fibrinogen, or fibrin layers were subsequently immobilized by physisorption or by covalent coupling using imidoester chemistry. Immobilization of all the tested proteins resulted in significantly enhanced cell adhesion on these polymers. Moreover, we found that covalently immobilized proteins supported significantly greater cell proliferation than physisorbed proteins over 6 days. SMC cultured on P(100/0) films modified by covalently attached fibronectin or fibrin layers proliferated at a rate comparable to that observed on control tissue culture polystyrene. The proposed surface modification schemes were much less efficient in improving cell attachment and proliferation on P(70/30) films. However, prewetting P(70/30) with a phosphate buffer prior to aminolysis significantly improved cell numbers following immobilization of fibronectin. Immunostaining of smooth muscle-specific alpha-actin of SMC grown on protein-modified P(100/0) 8 h and 48 h after cell seeding, confirmed preserved SMC phenotype on all modified surfaces.

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“…The pH profile of the immobilized glucoamylase was not significantly different from that of the native enzyme [181]. A few examples have, however, shown that enzymes coupled to carriers via diimidate esters usually had higher activity and stability [456]. Although it has been observed that immobilization of enzymes on to the carriers bearing terminal amino groups at the end of a long spacer results in better retention of activity [125], other studies have revealed that activation of carriers bearing carboxylic functionality, then attachment of the enzyme through the amino group to the activated carrier bearing carboxylic groups led to formation of more stable immobilized enzymes.…”

Section: Polymers Containing Amino Groupsmentioning

confidence: 99%

“…Conversion to Carboxyl Groups As discussed above, immobilization of enzymes on a carrier bearing amino groups by peptide formation can be realized by attaching the activated enzyme with carbodiimide methods [372]. For instance, protein kinase CK2 and calf intestine alkaline phosphatase CIP immobilized on a resin bearing secondary amino and thiol groups via the imidoester di-Me pimelimidate hydrochloride had constant storage stability and activity for at least 3 months [456]. Thus, it is necessary to convert the amino functionality into carboxyl groups.…”

Section: Polymers Containing Amino Groupsmentioning

confidence: 99%

Covalent Enzyme Immobilization

Cao¹

2005

Carrier‐bound Immobilized Enzymes

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A Method of Immobilization on the Solid Support of Complex and Simple Enzymes Retaining Their Activity

Cited by 8 publications

References 20 publications

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Enhanced smooth muscle cell adhesion and proliferation on protein‐modified polycaprolactone‐based copolymers

Covalent Enzyme Immobilization

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