Serap Evran

The covalent bioconjugate of multiwalled carbon nanotube and amino-modified linearized plasmid DNA for gene delivery

Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well‐known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC). The whole pQE‐70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6‐(6‐Amino)hexyl‐2′‐deoxyadenosine‐5′‐triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino‐modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat‐shock step at 42°C. The presence of Ca2+ in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

Directed evolution of (βα)8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase α-subunit

Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase α-subunit (TrpA) are (βα)(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation.

Modification of Porcine Pancreatic Lipase with Z‐Proline

Abstract Porcine pancreatic lipase was modified with Z‐proline via the constitution of amide bonds between the free amino groups of lipase and the carboxyl groups of Z‐proline, which were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC). Different amounts of Z‐proline were bound to lipase. Modification degree was determined by 2,4,6‐trinitrobenzene sulphonic acid (TNBS), by means of the decrease in free amino groups on lipase. The reason for choosing Z‐proline was its unique structural characteristics, protected amino groups, and its effect on protein conformation by reducing the flexibility of the lipase molecule, thus achieving stabilization against changes in pH and temperature. After the modification, porcine pancreatic lipase was found to have new physicochemical characteristics, such as optimum alkaline pH stability and thermal stability at elevated temperatures.

Site-directed mutagenesis of methionine residues for improving the oxidative stability of α-amylase from Thermotoga maritima.

The oxidative stability of α-amylase (AmyC) from Thermotoga maritima was improved by mutating the methionine residues at positions 43 and 44, 55, and 62 to oxidative-resistant alanine residues. The most resistant M55A variant showed 50% residual activity in the presence of 100 mM H₂O₂, whereas the wild-type enzyme was inactive.

Photoinduced in situ formation of clickable PEG hydrogels and their antibody conjugation

A simple approach for the preparation of a clickable poly(ethylene glycol)-based hydrogel as a polymeric support for protein immobilization via photoinitiated free radical polymerization of poly(ethylene glycol) diacrylate and propargyl acrylate is established. Bioconjugation to the obtained gels was achieved by azide–alkyne click reaction with azido-functionalized anti-immunoglobulin G (anti-IgG) and anti-His tag antibodies. Evaluation of the affinities of the PEG hydrogels to the corresponding substrates (IgG and His-tagged YFP, respectively) indicates their specific binding capability.

Improving thermal and detergent stability of Bacillus stearothermophilus neopullulanase by rational enzyme design

Neopullulanase, a glycosyl hydrolase from Bacillus stearothermophilus (bsNpl), is a potentially valuable enzyme for starch and detergent industries. However, as the protein is not active at elevated temperatures and high surfactant concentrations, we aimed to increase its stability by rational enzyme design. Nine potentially destabilizing cavities were identified in the crystal structure of the enzyme. Based on computational predictions, these cavities were filled by residues with bulkier side chains. The five Asp46Glu, Val239Leu, Val404Leu, Ser407Thr and Ala566Leu exchanges resulted in a drastic stabilization of bsNpl against inactivation by heat and detergents. The catalytic activity of the variants was identical to the wild-type enzyme.

DEVELOPMENT OF GENETICALLY ENCODED FLUORESCENT PROTEIN CONSTRUCTS OF HYPERTHERMOPHILIC MALTOSE-BINDING PROTEIN

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.

Isolation and Immobilization of His-Tagged Alcohol Dehydrogenase on Magnetic Nanoparticles in One Step: Application as Biosensor Platform

His-tagged Alcohol dehydrogenase was produced as a recombinant protein in E. coli. Afterwards, isolation and immobilization of the enzyme was carried in one-step via copper modified magnetic nanoparticles (MNPs) by the effect of interactions between Cu and histidine. The resulting enzyme bound MNPs was then attached to the surface of carbon paste electrode by the magnetic force and used as an electrochemical biosensor for the alcohol sensing applications.

Cell-penetrating peptides in nanodelivery of nucleic acids and drugs

Abstract The hydrophobic nature of cell membranes is one of the major obstacles in the therapeutic delivery of nucleic acids and drug-loaded nanoparticles. Cell-penetrating peptides (CPPs) have the ability to pass biological membranes and enter cells. Due to this intrinsic property, CPPs are employed as vectors for intracellular delivery of nucleic acids and nanoparticles. In this chapter, we first briefly describe the classification and uptake mechanisms of CPPs. Then, we describe the recent therapeutic applications of CPP-modified nanoparticles as drug carriers. In this context, we give an overview of covalent and noncovalent conjugation of CPPs. The second part involves the use of CPPs in nonviral delivery of nucleic acids. Although viral vectors are highly efficient systems for introducing genes, the safety issues with viral systems need to be considered. Nanoparticle-based nonviral vectors provide an attractive alternative, but their gene transfection efficiency is very low. Therefore, novel design strategies are needed to enhance the efficiency. We summarize the use of CPPs in enhancing gene transfer efficiency of nonviral vectors. Besides the clinical potential of currently known CPPs, we also discuss the limitations and the need for designing novel CPPs.

Pathogen-specific nucleic acid aptamers as targeting components of antibiotic and gene delivery systems

Abstract Inefficient delivery of antibiotics to the infection microenvironment, side effects of high-dose antibiotic use, as well as antimicrobial drug resistance are major global issues that remain to be solved. Efficient drug delivery systems and novel antimicrobial treatment strategies are urgently needed. Cell-specific aptamers are excellent molecules for specific targeting of microbial cells. Aptamers are single-stranded DNA or RNA molecules, which are able to selectively bind to their targets with high affinity. In this chapter, we first describe the methods for in vitro selection of aptamers against the whole pathogenic cell. Then we present an overview of current cell-specific aptamer-conjugated nanoparticles. In addition, we address two issues: nanoparticle-based antimicrobial gene delivery and modification of nanocarriers with aptamers.