Hydrodynamic behavior and thermal stability of a PEGylated protein: Studies with hen egg lysozyme

We studied the effect of covalent attachment of polyethylene glycol (PEGylation) on the hydrodynamic behavior and thermal stability of a model protein---Hen Egg Lysozyme (HEL). HEL was modified with a linear, 20-kD, PEG to produce mono (PEG1-HEL), di (PEG2-HEL), and triPEGylated (PEG3-HEL) species. The hydrodynamic properties of HEL were altered upon PEGylation. A decrease in sedimentation (s) and diffusion (D) coefficients was observed for all three PEG-HEL molecules in comparison to HEL (1.81 s). Despite differences in molecular weights of the PEG-HEL molecules (∼34, 55 and 80 kD), their s values were very close (1.0--1.1 s). Significant hydrodynamic non-ideality was observed for the PEG-HEL molecules, however, their Stokes radii (Rh) calculated from Do1 values were in agreement with dynamic light scattering (DLS) measurements. The Rh of HEL increased dramatically from 20 A to ∼50 A upon modification with a single 20-kD PEG chain. PEG2-HEL and PEG3-HEL had even larger radii of ∼68 A and 74 A. DLS studies with various PEGS (MW 5000--40,000) indicated that PEG is a random coil in solution. The Rh of PEG1-HEL and PEG2-HEL were measured to be only ∼10% larger than the 20-kD (43 A) and 40-kD (60 A) PEG chains. These data suggest that the covalently tethered PEG(s) predominantly govern the solution conformation of the PEG-HEL molecules. The thermal stability of PEGylated HEL was evaluated by employing the Eyring-Lumry model ( N↔TmD →kaA )2 for protein aggregation. A decrease in the melting temperature (Tm) of HEL unfolding was observed with increasing degree of PEGylation, which is indicative of thermodynamic instability. A Tm drop of up to 2.5°C (DSC) and 4.0°C (Difference Spectrum method) was observed for the PEG-HEL molecules. In contrast, turbidimetric studies showed that the kinetic aggregation rate (ka) of the PEG-HEL molecules was dramatically lower in comparison to the native HEL. Size exclusion HPLC indicated that the extent of aggregation decreased with increasing degree of PEGylation; only 34% of the HEL monomer remained after incubation at 75°C for 30 minutes, while 68% and 79% of the PEG1-HEL and PEG2-HEL monomers were present. These data suggest that the thermal stability of PEGylated HEL is kinetically controlled. The Tm may not be a true indicator of the stability of a PEG-protein with respect to aggregation. 1Do---Diffusion coefficient value extrapolated to infinite dilution. 2N---native state, D---denatured state, A---aggregate

Particle Shape Enhances Specificity of Antibody-Displaying Nanoparticles

Monoclonal antibodies are used in numerous therapeutic and diagnostic applications; however, their efficacy is contingent on specificity and avidity. Here, we show that presentation of antibodies on the surface of nonspherical particles enhances antibody specificity as well as avidity toward their targets. Using spherical, rod-, and disk-shaped polystyrene nano- and microparticles and trastuzumab as the targeting antibody, we studied specific and nonspecific uptake in three breast cancer cell lines: BT-474, SK-BR-3, and MDA-MB-231. Rods exhibited higher specific uptake and lower nonspecific uptake in all cells compared with spheres. This surprising interplay between particle shape and antibodies originates from the unique role of shape in determining binding and unbinding of particles to cell surface. In addition to exhibiting higher binding and internalization, trastuzumab-coated rods also exhibited greater inhibition of BT-474 breast cancer cell growth in vitro to a level that could not be attained by soluble forms of the antibody. The effect of trastuzumab-coated rods on cells was enhanced further by replacing polystyrene particles with pure chemotherapeutic drug nanoparticles of comparable dimensions made from camptothecin. Trastuzumab-coated camptothecin nanoparticles inhibited cell growth at a dose 1,000-fold lower than that required for comparable inhibition of growth using soluble trastuzumab and 10-fold lower than that using BSA-coated camptothecin. These results open unique opportunities for particulate forms of antibodies in therapeutics and diagnostics

Mapping Site-Specific Changes that Affect Stability of the NTerminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the 1H-15N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability

Increased Hydrogen Production by Genetic Engineering of Escherichia coli

Escherichia coli is capable of producing hydrogen under anaerobic growth conditions. Formate is converted to hydrogen in the fermenting cell by the formate hydrogenlyase enzyme system. The specific hydrogen yield from glucose was improved by the modification of transcriptional regulators and metabolic enzymes involved in the dissimilation of pyruvate and formate. The engineered E. coli strains ZF1 (ΔfocA; disrupted in a formate transporter gene) and ZF3 (ΔnarL; disrupted in a global transcriptional regulator gene) produced 14.9, and 14.4 µmols of hydrogen/mg of dry cell weight, respectively, compared to 9.8 µmols of hydrogen/mg of dry cell weight generated by wild-type E. coli strain W3110. The molar yield of hydrogen for strain ZF3 was 0.96 mols of hydrogen/mol of glucose, compared to 0.54 mols of hydrogen/mol of glucose for the wild-type E. coli strain. The expression of the global transcriptional regulator protein FNR at levels above natural abundance had a synergistic effect on increasing the hydrogen yield in the ΔfocA genetic background. The modification of global transcriptional regulators to modulate the expression of multiple operons required for the biosynthesis of formate hydrogenlyase represents a practical approach to improve hydrogen production

OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis