Mehmet Inan

Progress in the development of a recombinant vaccine for human hookworm disease: The Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.

Non-Repressing Carbon Sources for Alcohol Oxidase (AOX1) Promoter of Pichia pastoris.

The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1 utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1 utilizing enzymes, especially in a shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress beta-gal production when methanol was used as an inducer in mut- strain of P. pastoris. Our results show that either one of alanine, sorbitol, mannitol or trehalose can be used as a sole carbon and energy source for P. pastoris, although the doubling time on trehalose was very long. Mut- strains growing in media containing trehalose, alanine, sorbitol and mannitol with methanol (0.5%) as an inducing agent expressed as much or higher amount of beta-gal as compared to the mut+ growing in methanol containing media.

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

Fermentation strategies for recombinant protein production inPichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

Optimization of temperature-glycerol-pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris.

Abstract This study was undertaken to determine the optimum pH, temperature and glycerol feed rate for the production of recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (rAcAP-5) by Pichia pastoris using response surface methodology (RSM). A central composite design was used as an experimental design for allocation of treatment combinations in three blocks. The variables selected for study were pH, temperature and glycerol feed rate. pH was the most important variable affecting yield, specific yield and specific activity of rAcAP-5. Glycerol feed rate had a significant effect on the specific activity of rAcAP-5 (% of total secreted protein) while temperature did not have a significant effect on the responses. The data showed a trend that gave maximum responses and there was no blocking effect on the responses. The RSM formulated three second order polynomial empirical models relating to the responses. From these models it was possible to determine the optimum conditions variables for maximum yield of rAcAP-5 (1.2 g l−1), the maximum specific yield of rAcAP-5 (11.5 mg g−1 dry cell) and the maximum specific activity of rAcAP-5 (96% of total secreted protein).

Maximization of Production of Secreted Recombinant Proteins in Pichia pastoris Fed‐Batch Fermentation

Pontryaginapos;s Maximum Principle has been applied for optimization of secreted proteins from Pichia pastoris fed‐batch fermentation. The objective of this work is to maximize the total accumulated product per unit operation time under different given conditions and system constraints. To obtain optimal solutions, an automated curve‐fitting software, Table Curve 2D, was employed to construct the necessary mathematical models and solve the complicated functions. In the solution processes, the end of the glycerol batch phase was defined as the initial state of the system, the end of the methanol fed‐batch phase as the final state, the cell mass produced along with product accumulated as state variables, and the specific growth rate (μ) as the control variable. Initially, a relationship between the specific production rate (ρ) and μ was established. Then, according to Pontryaginapos;s Maximum Principle, the admissible range of μ and its trajectories for the optimal operations were determined. Four representative cases with different combinations of the operation time along with the initial and final states were evaluated. A close correlation was obtained between the predicted values of the model equation with the experimental results from the Pichia pastoris fed‐batch fermentations producing secreted α‐galactosidase. The approaches proposed here greatly simplify the computational processes and validate the optimization strategy as a generalized approach to maximize the yield from fed‐batch fermentations.

The Effect of Ethanol and Acetate on Protein Expression in Pichia pastoris.

Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mut+, Mut(s), or Mut-). We have nvestigated ethanol repression of the AOX1 promoter using strains, GS115 (Mut+) and MC100-3 (Mut-), expressing an AOX1-lacZ fusion. The addition of 10 mg l(-1) ethanol at the start of methanol induction delayed beta-galactosidase production and methanol utilization for four hours in shake flask experiments. When ethanol and acetate were added together, all of the ethanol was converted to acetate, which also represses the AOX1 promoter. The effects of ethanol and acetate on protein expression in P. pastoris at shake flask and fermentor conditions are discussed.

Self-assembled nanoparticle necklaces network showing single-electron switching at room temperature and biogating current by living microorganisms.

A network of one-dimensional (1D) Au nanoparticle necklaces is synthesized and shown to exhibit electronic switching, that is, gating, by the metabolic activity of yeast cells deposited on the structure. Without the cells, the network exhibits the Coulomb blockade effect at room temperature with a sharp threshold voltage, V(T) of approximately 0.45 V, which corresponds to a switching energy of approximately 20 kT. Although the enhancement in V(T) from approximately 70 mV for a single (10 nm) Au particle to >1 V is well-known for a 2D array, the uniqueness of the network topology is the relatively weak dependence of V(T) on temperature that leads to room temperature switching behavior, in contrast to an array where the blockade effect vanishes at ambient temperatures. The coupling between the biochemical process of the cell and the electronics of the network has potential applications for making electrodes for biofuel cells and highly sensitive biosensors using the cell as the specific sensing moiety.

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate

BackgroundYeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.ResultsHere, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man5GlcNAc2 N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.ConclusionsTogether, these data illustrate that the GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins.

Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus.

Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 A have been collected from a crystal that belongs to the monoclinic space group P2(1) with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 A, beta = 112.12 degrees. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit.

Rational Design and Optimization of Fed-Batch and Continuous Fermentations

This chapter provides rational approaches to design and optimize fed-batch and continuous fermentations of both Mut+ and Muts (methanol utilization plus and slow) Pichia pastoris strains. The methods are described in detail for glycerol batch, glycerol fed-batch, transition, and methanol fed-batch/mixed feed/ continuous stirred tank reactor (CSTR) phases of the process based on glycerol and methanol consumption models. Cell density, broth volume, substrate feed rate, and the length of each phase are rationally designed to conduct runs with selected parameters for optimizing a process. The optimization is anchored by the impact of a specific growth rate/dilution time (for CSTRs) on productivity. Equations for simulation of a process with optimal parameters are derived for an optimal process design. This protocol can be used as a practical manual for process development of a P. pastoris recombinant fermentation, and also as a reference for fermentation of other microorganisms.