Chenming Zhang

Tobacco protein separation by aqueous two-phase extraction

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.

Study and design of stability in GH5 cellulases

Thermostable enzymes that hydrolyze lignocellulosic materials provide potential advantages in process configuration and enhancement of production efficiency over their mesophilic counterparts in the bioethanol industry. In this study, the dynamics of β‐1,4‐endoglucanases (EC: 3.2.1.4) from family 5 of glycoside hydrolases (GH5) were investigated computationally. The conformational flexibility of 12 GH5 cellulases, ranging from psychrophilic to hyperthermophilic, was investigated by molecular dynamics (MD) simulations at elevated temperatures. The results indicated that the protein flexibility and optimum activity temperatures are appreciably correlated. Intra‐protein interactions, packing density and solvent accessible area were further examined in crystal structures to investigate factors that are possibly involved in higher rigidity of thermostable cellulases. The MD simulations and the rules learned from analyses of stabilizing factors were used in design of mutations toward the thermostabilization of cellulase C, one of the GH5 endoglucanases. This enzyme was successfully stabilized both chemically and thermally by introduction of a new disulfide cross‐link to its highly mobile 56‐amino acid subdomain. Biotechnol. Bioeng. 2012;109: 31–44.

Natural product-based nanomedicine: recent advances and issues

Natural products have been used in medicine for many years. Many top-selling pharmaceuticals are natural compounds or their derivatives. These plant- or microorganism-derived compounds have shown potential as therapeutic agents against cancer, microbial infection, inflammation, and other disease conditions. However, their success in clinical trials has been less impressive, partly due to the compounds’ low bioavailability. The incorporation of nanoparticles into a delivery system for natural products would be a major advance in the efforts to increase their therapeutic effects. Recently, advances have been made showing that nanoparticles can significantly increase the bioavailability of natural products both in vitro and in vivo. Nanotechnology has demonstrated its capability to manipulate particles in order to target specific areas of the body and control the release of drugs. Although there are many benefits to applying nanotechnology for better delivery of natural products, it is not without issues. Drug targeting remains a challenge and potential nanoparticle toxicity needs to be further investigated, especially if these systems are to be used to treat chronic human diseases. This review aims to summarize recent progress in several key areas relevant to natural products in nanoparticle delivery systems for biomedical applications.

Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Currently, killed-virus and modified-live PRRSV vaccines are used to control porcine reproductive and respiratory syndrome disease (PRRS). However, very limited efficacy of killed-virus vaccines and serious safety concerns for modified-live virus vaccines demand the development of novel PRRSV vaccines. In this report, we investigated the possibility of using transgenic plants as a cost-effective and scalable system for production and delivery of a viral protein as an oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-γ response was also detected in splenocytes of vaccinated animals. These results demonstrate that transgenic corn plants are an efficient subunit vaccine production and oral delivery system for generation of both systemic and mucosal immune responses against PRRSV.

A novel and efficient nicotine vaccine using nano-lipoplex as a delivery vehicle

A number of vaccines conjugating nicotine haptens with carrier proteins have been developed to combat nicotine caused tobacco dependence. Some vaccines, such as NicVAX, NicQb, advanced into clinical trials, but none of them were successful. Most of those vaccines have some innate disadvantages such as low nicotine loading capacity, easy degradation, and vulnerable to the clearance by reticulo-endothelial system (RES). Thus, there is undoubtedly an urgent need for developing novel vaccines against nicotine addiction. In this study, we assembled a liposome-protein based nanoparticle as a nicotine hapten delivery system. The nanoparticle (Scheme 1) was constructed by conjugating a model hapten carrier protein, bovine serum albumin (BSA), to cationic liposomes. This nano-sized complex, lipoplex, was characterized using zetasizer, transmission electron microscope (TEM), and flow cytometry. The efficacy of the lipoplex vaccine was evaluated in mice and compared with that of Nicotine-BSA conjugate (Nic-BSA). The lipoplex vaccine with Alum was able to elicit the highest NicAb titer of 11169 ± 2112, which was significantly higher than that induced by either the vaccine without Alum or Nic-BSA with Alum. The significant immunostimulatory effect of this nano-lipoplex may provide a novel strategy to improve the immunogenic ability of current nicotine vaccines or other vaccines using small molecules as immunogens.

In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters

Due to the many beneficial properties combined from both poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and liposomes, lipid-PLGA hybrid NPs have been intensively studied as cancer drug delivery systems, bio-imaging agent carriers, as well as antigen delivery vehicles. However, the impact of lipid composition on the performance of lipid-PLGA hybrid NPs as a delivery system has not been well investigated. In this study, the influence of lipid composition on the stability of the hybrid NPs and in vitro antigen release from NPs under different conditions was examined. The uptake of hybrid NPs with various surface charges by dendritic cells (DCs) was carefully studied. The results showed that PLGA NPs enveloped by a lipid shell with more positive surface charges could improve the stability of the hybrid NPs, enable better controlled release of antigens encapsulated in PLGA NPs, as well as enhance uptake of NPs by DC.

Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DPu2009=u200914) and more than 50% on the long-chain RAC (DPu2009=u2009164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).

Engineering a large protein by combined rational and random approaches: stabilizing the Clostridium thermocellum cellobiose phosphorylase

The Clostridium thermocellum cellobiose phosphorylase (CtCBP) is a large protein consisting of 812 amino acids and has great potential in the production of sugar phosphates, novel glycosides, and biofuels. It is relatively stable at 50 °C, but is rapidly inactivated at 70 °C. To stabilize CtCBP at elevated temperatures, two protein-engineering approaches were applied, i.e. site-directed mutagenesis based on structure-guided homology analysis and random mutagenesis at various mutation rates. The former chose substitutions by comparison of the protein sequences of CBP homologs, utilized structural information to identify key amino acid residues responsible for enhanced stability, and then created a few variants accurately. The latter constructed large libraries of random mutants at different mutagenesis frequencies. A novel combinational selection/screening strategy was employed to quickly isolate thermostability-enhanced and active variants. Several stability-enhanced mutants were obtained by both methods. Manually combining the stabilizing mutations identified from both rational and random approaches led to the best mutant (CM3) with the halftime of inactivation at 70 °C extended from 8.3 to 24.6 min. The temperature optimum of CM3 was increased from 60 to 80 °C. These results suggested that a combination of rational design and random mutagenesis could have a solid basis for engineering large proteins.

Probing the Active Site Chemistry of β‑Glucosidases along the Hydrolysis Reaction Pathway

β-Glucosidases (EC 3.2.1.21) can be found in all domains of living organisms, where they play essential roles in the removal of terminal glucosyl residues from nonreducing ends of saccharides and glycosides. Two active site amino acid residues, a nucleophile and a proton donor, play key roles in the hydrolytic mechanism. Besides these two highly conserved catalytic residues, there are other conserved amino acids in the active site of β-glucosidases that make direct hydrogen bonds to the glycosyl moiety at the -1 subsite. In this study, the catalytic mechanism of a GH1 β-glucosidase (BGlu1) is systematically studied. On the basis of the quantum mechanical studies, the side chain of Tyr315 in an interaction with both O5 of the glucose ring and the nucleophilic glutamate contributes significantly to the energy profile. Glu440 and the conserved Asn175 are the other residues in the polar interaction with -1 glucose with considerable influence on the free energy of the reaction. Gln29, His130, and Trp441, which also form hydrogen bonds to the glycosyl moiety, are found to have relatively a minor effect on the reaction. Different arrangements of active site residues in the high-level [quantum mechanics (QM)] and low-level [molecular mechanics (MM)] regions during the hybrid QM/MM calculations indicate that Tyr315 lowers the energy barrier in the deglycosylation step (by 11.95 kcal/mol) while Glu440 mainly reduces the energy barrier of the glycosylation step. Exclusion of either of these two residues from the QM region results in deviation of the geometric parameters of the enzyme-substrate complex from those expected for the preactivated distorted structure of the substrate.

Assembly of single-walled carbon nanohorn supported liposome particles.

Nanoparticle-supported liposomes can be a promising platform for drug delivery, vaccine development, and biomedical imaging. Single-walled carbon nanohorns are a relatively new carbon nanomaterial, and they could be used as carriers of drug and imaging reagents. Assembling lipids around carbon nanohorns would confer this nanomaterial much broader applications such as vaccine development and targeted drug delivery by embedding a target protein or immunogenic protein into the lipid bilayer structure. Here, we show the assembly of functionalized single-walled carbon nanohorns (-CH(2)-CH(2)-COOH(x), ~100 nm) with positively charged lipids through a freeze and thaw cycle. The assembled complex particles can be readily separated from individual nanohorns or liposomes under specific centrifugation conditions. The results from transmission electronic microscopy, flow cytometry through nitrobenzoxadiazole labeled lipids, and zeta potential analysis clearly show that the nanohorns are encapsulated by liposomes with a median size of ca. 120 nm.