Reliable microfluidic on-chip incubation of droplets in delay-lines.
AuthorsFrenz L, Blank K, Brouzes E, Griffiths AD,
JournalLab Chip
PubMed ID19417899
Together with droplet creation, fusion and sorting, the incubation of droplets is one of the most important and essential operations for droplet-based microfluidic assays. This manuscript concerns the development of delay-lines, which are necessary to allow incubation of reactions for precise time periods. We analyze the problems associated with creating ... More
A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters.
AuthorsPorter JR, Stains CI, Jester BW, Ghosh I,
JournalJ Am Chem Soc
PubMed ID18444624
Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems ... More
Novel beta-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy.
AuthorsShukla GS, Krag DN,
JournalJ Drug Target
PubMed ID19751096
'Novel phage-displayed random linear dodecapeptide (X(12)) and cysteine-constrained decapeptide (CX(10)C) libraries constructed in fusion to the amino-terminus of P99 beta-lactamase molecules were used for identifying beta-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round ... More
Fluorogenic cephalosporin substrates for ß-lactamase TEM-1.
AuthorsRukavishnikov A, Gee KR, Johnson I, Corry S,
JournalAnal Biochem
PubMed ID21867672
'Cephalosporin was used to synthesize soluble and precipitating fluorogenic ß-lactam substrates that demonstrated differential catalytic hydrolysis by three different subtypes of ß-lactamase: TEM-1 (class A), p99 (class C), and a Bacillus cereus enzyme sold by Genzyme (class B). The most successful soluble substrate contained difluorofluorescein (Oregon Green 488) ligated to ... More
Imaging tuberculosis with endogenous beta-lactamase reporter enzyme fluorescence in live mice.
AuthorsKong Y, Yao H, Ren H, Subbian S, Cirillo SL, Sacchettini JC, Rao J, Cirillo JD,
JournalProc Natl Acad Sci U S A
PubMed ID20566877
'The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, ... More
Activation of circularly permutated ß-lactamase tethered to antibody domains by specific small molecules.
AuthorsKojima M, Iwai H, Dong J, Lim SL, Ito S, Okumura K, Ihara M, Ueda H,
JournalBioconjug Chem
PubMed ID21446744
Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv ... More
An autoinhibited coiled-coil design strategy for split-protein protease sensors.
AuthorsShekhawat SS, Porter JR, Sriprasad A, Ghosh I,
JournalJ Am Chem Soc
PubMed ID19803505
Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of ... More
Developing bifunctional beta-lactamase molecules with built-in target-recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage-display selection.
AuthorsShukla GS, Krag DN,
JournalJ Mol Recognit
PubMed ID19437416
This study was focused on developing catalytically active beta-lactamase enzyme molecules that have target-recognizing sites built within their scaffold. Using phage-display approach, nine libraries were constructed by inserting the randomized linear or cysteine-constrained heptapeptides in the five different loops on the outer surface of P99 beta-lactamase molecule. The pIII signal ... More
Cancer cell-specific internalizing ligands from phage displayed beta-lactamase-peptide fusion libraries.
AuthorsShukla GS, Krag DN,
JournalProtein Eng Des Sel
PubMed ID20219829
The present study was focused on identifying cancer cell-specific internalizing ligands using a new kind of phage display library in which the linear or cysteine-constrained random peptides were at amino-terminus fusion to catalytically active P99 beta-lactamase molecules. The size and quality of libraries were comparable to other reported phage display ... More
Phage-displayed combinatorial peptide libraries in fusion to beta-lactamase as reporter for an accelerated clone screening: Potential uses of selected enzyme-linked affinity reagents in downstream applications.
AuthorsShukla GS, Krag DN,
JournalComb Chem High Throughput Screen
PubMed ID20214576
Phage-display selection of combinatorial libraries is a powerful technique for identifying binding ligands against desired targets. Evaluation of target binding capacity of multiple clones recovered from phage display selection to a specific target is laborious, time-consuming, and a rate-limiting step. We constructed phage-display combinatorial peptide libraries in fusion with a ... More
Activation of cyclin-dependent kinase 5 by calpains contributes to human immunodeficiency virus-induced neurotoxicity.
AuthorsWang Y, White MG, Akay C, Chodroff RA, Robinson J, Lindl KA, Dichter MA, Qian Y, Mao Z, Kolson DL, Jordan-Sciutto KL
JournalJ Neurochem
PubMed ID17897354
Although the specific mechanism of neuronal damage in human immunodeficiency virus (HIV) -associated dementia is not known, a prominent role for NMDA receptor (NMDAR)-induced excitotoxicity has been demonstrated in neurons exposed to HIV-infected/activated macrophages. We hypothesized NMDAR-mediated activation of the calcium-dependent protease, calpain, would contribute to cell death by induction ... More