A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (η) of annealing as a function of temperature (TA), annealing period tA) and template composition. The optimal efficiency lies in a narrow region of TA and tA for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX, and HBB (with 78.72% and 52.99% GC respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3 to 6 seconds and depend on annealing temperature. Annealing times greater than 10 seconds yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that, shorter annealing times are not only sufficient but also necessary for efficient PCR amplification of GC-rich templates.

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes.

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

SummaryPhosphoenolpyruvate carboxylase (PEPC [Ppc]) has been previously purified and characterized in biochemical and immunological terms from two green microalgae, Chlamydomonas reinhardtii and Selenastrum minutum. The findings indicate that these algae possess at least two distinct PEPC enzyme-forms, homotetrameric Class-1 and heteromeric Class-2, that differ significantly from each other and their plant and prokaryotic counterparts. Surprisingly, however, green-algal PEPC has been unexplored to date in molecular terms. This study reports the molecular cloning of the two Ppc genes in C. reinhardtii (CrPpc1, CrPpc2), each of which is transcribed in vivo and encodes a fully active, recombinant PEPC that lacks the regulatory, N-terminal serylphosphorylation domain that typifies the vascular-plant enzyme. These distinct catalytic subunit-types differ with respect to their (i) predicted molecular mass ($108.9 [CrPpc1] versus $131.2 kDa [CrPpc2]) and critical C-terminal tetrapeptide; and (ii) immunoreactivity with antisera against the p102 and p130 polypeptides of S. minutum PEPC1/PEPC2 and PEPC2, respectively. Only the Ppc1 transcript encodes the p102 catalytic subunits common to both Class-1 and Class-2 enzyme-forms in C. reinhardtii. The steady-state transcript levels of both CrPpc1/2 are coordinately up-/down-regulated by changes in [CO 2 ] or [NH þ 4 ] during growth, and generally mirror the response of cytoplasmic glutamine synthetase (Gs1) transcript abundance to changes in inorganic [N] at 5% CO 2 . These collective findings provide key molecular insight into the Ppc genes and corresponding PEPC catalytic subunits in the eukaryotic algae.

Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.

SummaryApplication of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48 ⁄ 45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plantproduced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48 ⁄ 45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.

Chondrocytes, the cellular component of the articular cartilage, have long been recognized as strain-sensitive cells, and have the ability to sense mechanical stimulation through surface receptors and intracellular signaling pathways. This strain-induced biological response of chondrocytes has been exploited to facilitate chondrocyte culture in in vitro systems; examples include the application of hydrostatic pressure, dynamic compression, hydrodynamic shear (i.e., rotating bioreactors), and low-intensity pulsed ultrasound (US). While the ability of US to influence chondrogenesis has been documented, the precise mechanisms of US-induced stimulation continue to be investigated. There remains a critical need to evaluate the impact of US on chondrocytes in 3D culture, which is a necessary microenvironment for maintaining the chondrocyte phenotype. In this study, a continuous US wave for predetermined time intervals was employed, as opposed to pulsed US used in previous studies, to stimulate chondrocytes seeded in 3D scaffolds. The chondrocytes (n = 6) were subjected to US stimulation as follows: 1.5 MHz for 161 seconds, 5.0 MHz for 51 seconds, and 8.5 MHz for 24 seconds, and the US signal was applied twice in a 24-hour period. Scaffolds that are not stimulated by US served as the control. Both the control and the US-stimulated groups were maintained in culture for 10 days, and at the conclusion of the culture period, chondrocytes were assayed for total DNA content, morphology, and cartilage-specific gene expression by reverse transcriptase polymerase chain reaction. Our results show that chondrocytes when stimulated with continuous US for predetermined time intervals possessed higher cellular viability (1.2 to 1.4 times) and higher levels of type II collagen and aggrecan mRNA expression when compared to controls.

Phosphoglycolate phosphatase (PGPase), a key enzyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The enzyme was an ϳ65-kDa homodimer with a pI value of 5.1 composed of ϳ32-kDa subunits not connected by any S-S bridges. It was also highly specific for phosphoglycolate with a K m value of 140 M and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl 2 , and it recovered competitively following the addition of MgCl 2 or EGTA. A mobility shift was observed in SDSpolyacrylamide gel electrophoresis by the addition of CaCl 2 , indicating that the enzyme binds to Ca 2؉ . The N-terminal region of amino acid sequence deduced from cDNA sequence that was not contained in the purified PGPase had similar characteristics to those of typical stroma-targeting transit peptides in C. reinhardtii. The following region of the deduced sequence containing 302 amino acid residues was similar to p-nitrophenylphosphatase-like proteins, although the purified PGPase did not hydrolyze p-nitrophenylphosphate. Genomic DNA fragments from wild type containing the sequence homologous to the cDNA for PGPase complemented the PGPase-deficient mutant pgp1. Possible regulatory mechanisms during adaptation to limiting CO 2 were discussed based on the characteristics of the purified PGPase and the deduced amino acid sequence.