Charlene M. Mello

Purification and characterization of recombinant spider silk expressed in Escherichia coli

Abstract A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk.

Orientation difference of chemically immobilized and physically adsorbed biological molecules on polymers detected at the solid/liquid interfaces in situ

A surface sensitive second order nonlinear optical technique, sum frequency generation vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy. Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, for example, air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation, and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities.

Binding, inactivation, and adhesion forces between antimicrobial peptide cecropin P1 and pathogenic E. coli

The antimicrobial peptide cecropin P1 (CP1) exhibits broad spectrum activity against planktonic bacteria, including Escherichia coli (E. coli). However, its activity when attached to a substrate has not been thoroughly studied. We immobilized CP1 to gold or silicon nitride, and studied how the method of attachment of peptide to the surface affected peptide interaction with and killing of the bacteria. Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we characterized non-specific binding between CP1 to silicon nitride and gold, and covalent binding of cysteine-terminated CP1 (CP1-cys) to gold. The density of CP1-cys adsorbed on gold was more than the density of CP1 on silicon nitride, and activity against E. coli also depended on the method of attachment used to anchor the peptide to the surface. Twelve E. coli strains with known lipopolysaccharide (LPS) structures were studied. Bacterial adhesion with CP1 was strongest for E. coli with long O-antigens, as determined by atomic force microscopy (AFM). This may be caused by CP1 interacting with the hydrophilic part of the LPS, while control bacteria or those with short O-antigens had their hydrophobic lipid A region more exposed. Killing of E. coli due to contact with CP1 was dependent on the method by which the peptide was immobilized. Four out of 12 E. coli strains were killed when contacted with CP1-cys bound to gold via a thiol bond, while all 12 strains could be killed when in contact with CP1 on silicon nitride. In summary, both QCM-D adsorption experiments and adhesion forces measured by AFM showed a relationship between bacteria LPS length and binding or interaction with the antimicrobial peptide, but killing of E. coli by the peptide was most strongly dependent on how the peptide was attached to the surface.

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.

Biosynthesis and Processing of Silk Proteins

Silks produced by silkworms (e.g., Bombyx mori ) and orb-web weaving spiders (e.g., Nephila clavipes ) are essentially pure protein, that is, complexes of amino acid polymers. They are the most common fibers spun by biological systems. There has been a long-standing interest in the use of these and similar fibers in textiles, cables, fiber reinforcement in composites, in addition, for example, to cross hairs in optical instruments, and fishing nets. Both nylon, a homo-polymer of the amino acid glycine, and Kevlar, a polymer of a nonnatural aromatic amino acid, can be considered modified, synthetic versions of silk and are used for some of the applications mentioned above. The potential for genetic manipulation, through recombinant DNA technology, of the natural biosynthetic process for these natural proteins (see the article by Cappello in this issue) has renewed interest in the production of new silklike proteins. The natural silks are characterized by a β -sheet secondary structure which is stabilized by interchain hydrogen bonds and intersheet hydrophobic interactions (Figure 1). Silks can be considered block copolymers, with crystalline domains consisting of short side chain monomers (the amino acids glycine, alanine, and serine) interspersed in amorphous domains consisting of bulkier side chain amino acids. This family of fibers is naturally tailored to perform functions such as catching prey (orb web) or serving as a barrier against environmental challenges (cocoon). The domestic silkworm ( B. mori ) produces only one type of silk, cocoon silk, at only one stage in its lifecyle, during the fifth larval instar just before molt to the pupa. The silk is produced in modified salivary glands and spun from the mouth.

Solvent Effect and Time-Dependent Behavior of C-Terminus-Cysteine-Modified Cecropin P1 Chemically Immobilized on a Polymer Surface

Sum frequency generation (SFG) vibrational spectroscopy has been applied to the investigation of peptide immobilization on a polymer surface as a function of time and peptide conformation. Surface immobilization of biological molecules is important in many applications such as biosensors, antimicrobial materials, biobased fuel cells, nanofabrication, and multifunctional materials. Using C-terminus-cysteine-modified cecropin P1 (CP1c) as a model, we investigated the time-dependent immobilization behavior in situ in real time. In addition, potassium phosphate buffer (PB) and mixtures of PB and trifluoroethanol were utilized to examine the effect of peptide secondary structure on CP1c immobilization to polystyrene maleimide (PS-MA). The orientation of immobilized CP1c on PS-MA was determined using polarized SFG spectra. It was found that the peptide solution concentration, solvent composition, and assembly state (monomer vs dimer) prior to immobilization all influence the orientation of CP1c on a PS-MA surface. The detailed relationship between the interfacial peptide orientation and these immobilization conditions is discussed.

Detection and classification of related lipopolysaccharides via a small array of immobilized antimicrobial peptides.

A small array of antimicrobial peptides comprising three cysteine-terminated natural sequences covalently immobilized to pendant surface maleimide groups are used to bind and successfully discriminate five types of lipopolysaccharide (LPS) molecules. Using surface plasmon resonance, LPSs isolated from four strains of Escherichia coli and one strain of Pseudomonas aeruginosa yield distinct binding profiles to the three immobilized peptides. Linear discriminant analysis generated 100% training set and 80% validation set classification success for the 40 samples evaluated. This work demonstrates the discriminatory binding capabilities of immobilized antimicrobial peptides toward LPS molecules and alludes to their use as probes in pathogen sensing devices potentially superior to the current state-of-the-art.

Characterization of supported lipid bilayer disruption by chrysophsin-3 using QCM-D.

Antimicrobial peptides (AMPs) are naturally occurring polymers that can kill bacteria by destabilizing their membranes. A quartz crystal microbalance with dissipation monitoring (QCM-D) was used to better understand the action of the AMP chrysophsin-3 on supported lipid bilayers (SLB) of phosphatidylcholine. Interaction of the SLB with chrysophsin-3 at 0.05 μM demonstrated changes in frequency (Δf) and energy dissipation (ΔD) that were near zero, indicating little change in the membrane. At higher concentrations of chyrsophsin-3 (0.25-4 μM), decreases in Δf of up to 7 Hz were measured. These negative frequency changes suggest that mass was being added to the SLB, possibly due to peptide insertion into the membrane. At a chrysophsin-3 concentration of 10 μM, there was a net mass loss, which was attributed to pore formation in the membrane. QCM-D can be used to describe a mechanistic relationship between AMP concentration and interaction with a model cell membrane.

In Vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin

The category A agent, botulinum neurotoxin (BoNT), is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, we have identified three RNA aptamers through SELEX-process, which bind strongly to the light chain of type A BoNT (BoNT/A) and inhibit the endopeptidase activity, with IC(50) in low nM range. Inhibition kinetic studies reveal low nM K(I) and non-competitive nature of their inhibition. Aptamers are unique group of molecules as therapeutics, and this is first report of their development as an antidote against botulism. These data on K(I) and IC(50) strongly suggest that the aptamers have strong potential as antidotes that can reverse the symptom caused by BoNT/A.

The effects of solution structure on the surface conformation and orientation of a cysteine-terminated antimicrobial peptide cecropin P1

The surface structure of an antimicrobial peptide, cecropin P1, immobilized to a gold surface via a terminal cysteine residue was investigated. Using reflection-absorption infrared spectroscopy, surface plasmon resonance, and X-ray photoelectron spectroscopy, the effects of pH, solution conformation, and concentration on the immobilized peptide conformation, average orientation, and surface density were determined. Under all conditions investigated, the immobilized peptides were alpha-helical in a predominately flat, random orientation. The addition of the reducing agent Tris(2-carboxyethyl) phosphine hydrochloride to the buffer resulted in a twofold increase in immobilized peptide surface density.