SARS-CoV-2 spike protein S1 subunit induces potent neutralizing responses in mice and is effective against Delta and Omicron variants.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, belongs to the betacoronavirus genus. This virus has a high mutation rate, which rapidly evolves into new variants with different properties, such as increased transmissibility or immune evasion. Currently, the most prevalent global SARS-CoV-2 variant is Omicron, which is more transmissible than previous variants. Current available vaccines may be less effective against some currently existing SARS-CoV-2 variants, including the Omicron variant. The S1 subunit of the spike protein of SARS-CoV-2 has been a major target for COVID-19 vaccine development. It plays a crucial role in the virus's entry into host cells and is the primary target for neutralizing antibodies. In this study, the S1 subunit of the spike protein of SARS-CoV-2 was engineered and produced at a high level in Nicotiana benthamiana plant. The expression level of the recombinant S1 protein was greater than the 0.5-g/kg fresh weight, and the purification yield was at least ~0.3 g of pure protein/kg of plant biomass, which would make a plant-produced S1 antigen an ideal vaccine candidate for commercialization. Purified, the plant-produced SARS-CoV-2 S1 protein exhibited significantly higher binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Moreover, we also show that recombinant S1 protein/antigen-elicited antibodies can neutralize the Delta or Omicron variants. Collectively, our results demonstrate that a plant-produced S1 antigen could be a promising vaccine candidate against SARS-CoV-2 variants including Omicron.

Plant-produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel and highly pathogenic coronavirus that caused an outbreak in Wuhan City, China, in 2019 and then spread rapidly throughout the world. Although several coronavirus disease 2019 (COVID-19) vaccines are currently available for mass immunization, they are less effective against emerging SARS-CoV-2 variants, especially the Omicron (B.1.1.529). Recently, we successfully produced receptor-binding domain (RBD) variants of the spike (S) protein of SARS-CoV-2 and an antigen cocktail in Nicotiana benthamiana, which are highly produced in plants and elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. In this study, based on neutralization ability, we demonstrate that plant-produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants. These data demonstrate that plant-produced RBD and cocktail-based proteins are the most promising vaccine candidates and may protect against Delta and Omicron-mediated COVID-19. This is the first report describing vaccines against SARS-CoV-2, which demonstrate significant activities against Delta and Omicron variants.

Soluble Human Angiotensin- Converting Enzyme 2 as a Potential Therapeutic Tool for COVID-19 is Produced at High Levels In Nicotiana benthamiana Plant With Potent Anti-SARS-CoV-2 Activity.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs a safe, cost-effective SARS-CoV-2 vaccine as well as therapeutic and antiviral drugs to combat COVID-19. Angiotensin-converting enzyme 2 (ACE2), as a key receptor for SARS-CoV-2 infections, has been proposed as a potential therapeutic tool in patients with COVID-19. In this study, we report a high-level production (about ∼0.75 g/kg leaf biomass) of human soluble (truncated) ACE2 in the Nicotiana benthamiana plant. After the Ni-NTA single-step, the purification yields of recombinant plant produced ACE2 protein in glycosylated and deglycosylated forms calculated as ∼0.4 and 0.5 g/kg leaf biomass, respectively. The plant produced recombinant human soluble ACE2s successfully bind to the SARS-CoV-2 spike protein. Importantly, both deglycosylated and glycosylated forms of ACE2 are stable at increased temperatures for extended periods of time and demonstrated strong anti-SARS-CoV-2 activities in vitro. The half maximal inhibitory concentration (IC50) values of glycosylated ACE2 (gACE2) and deglycosylated ACE2 (dACE2) were ∼1.0 and 8.48 µg/ml, respectively, for the pre-entry infection, when incubated with 100TCID50 of SARS-CoV-2. Therefore, plant produced soluble ACE2s are promising cost-effective and safe candidates as a potential therapeutic tool in the treatment of patients with COVID-19.

Production and Characterization of Nucleocapsid and RBD Cocktail Antigens of SARS-CoV-2 in Nicotiana benthamiana Plant as a Vaccine Candidate against COVID-19.

The COVID-19 pandemic has put global public health at high risk, rapidly spreading around the world. Although several COVID-19 vaccines are available for mass immunization, the world still urgently needs highly effective, reliable, cost-effective, and safe SARS-CoV-2 coronavirus vaccines, as well as antiviral and therapeutic drugs, to control the COVID-19 pandemic given the emerging variant strains of the virus. Recently, we successfully produced receptor-binding domain (RBD) variants in the Nicotiana benthamiana plant as promising vaccine candidates against COVID-19 and demonstrated that mice immunized with these antigens elicited a high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2. In this study, we engineered the nucleocapsid (N) protein and co-expressed it with RBD of SARS-CoV-2 in Nicotiana benthamiana plant to produce an antigen cocktail. The purification yields were about 22 or 24 mg of pure protein/kg of plant biomass for N or N+RBD antigens, respectively. The purified plant produced N protein was recognized by N protein-specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant-produced N protein. In this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. Thus, obtained data support that a plant-produced antigen cocktail, developed in this study, is a promising vaccine candidate against COVID-19.

Plant-Produced Glycosylated and In Vivo Deglycosylated Receptor Binding Domain Proteins of SARS-CoV-2 Induce Potent Neutralizing Responses in Mice.

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.

A Plant-Produced in vivo deglycosylated full-length Pfs48/45 as a Transmission-Blocking Vaccine Candidate against malaria.

Pfs48/45 is a leading antigen candidate for a transmission blocking (TB) vaccine. However, efforts to produce affordable, safe and correctly folded full-length Pfs48/45 using different protein expression systems have not produced an antigen with satisfactory TB activity. Pfs48/45 has 16 cysteines involved in disulfide bond formation, and the correct formation is critical for proper folding and induction of TB antibodies. Moreover, Pfs48/45 is not a glycoprotein in the native hosts, but contains potential glycosylation sites, which are aberrantly glycosylated during expression in eukaryotic systems. Here, we demonstrate for the first time that full length, Endo H in vivo enzymatic deglycosylated Pfs48/45 antigen is produced at a high level in plants and is structurally stable at elevated temperatures. Sera from mice immunized with this antigen showed strong inhibition in SMFA. Thus, Endo H in vivo enzymatic deglycosylated Pfs48/45 is a promising candidate for the development of an affordable TB vaccine, which may have the potential to save millions.

Engineering, and production of functionally active human Furin in N. benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants.

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-ß-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

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-ß-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.

Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum.

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.

In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.

In vivo deglycosylation of recombinant proteins in plants by co-expression with bacterial PNGase F.

At present, several eukaryotic expression systems including yeast, insect and mammalian cells and plants are used for the production of recombinant proteins. Proteins with potential N-glycosylation sites are efficiently glycosylated when expressed in these systems. However, the ability of the eukaryotic expression systems to glycosylate may be not desirable for some proteins. If target proteins that do not carry N-linked glycans in the native host contain potential N-linked glycosylation sites, they can be aberrantly glycosylated in the eukaryotic expression systems, thus, potentially impairing biological activity. Recently, we have developed a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F via agroinfiltration followed by transient expression in plants. (1) Here, we summarize our work on this topic and its potential implications.

Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F.

Application 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 plant-produced 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.

The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds.

Both pulsed- and square-wave, low-intensity ultrasound (US) signals have been reported to impact chondrocyte function and biosynthetic activity. In this study, a low-intensity diffuse ultrasound (LIDUS) signal at 5.0 MHz (0.14 mW/cm(2)) was employed to stimulate bovine chondrocytes seeded in three-dimensional (3D) chitosan-based matrices. While the duration of application was constant at 51 s, US was applied once, twice, four times and eight times/day, and the impacts of US on the biosynthetic activity of chondrocytes and the expression of chondrocyte-specific genes were evaluated. When stimulated with continuous US for predetermined time intervals, chondrocytes had higher levels of type II collagen, aggrecan, L-Sox5 and Sox9 mRNA expression when compared to controls; however, under the same conditions, the expression of MMP-3 was downregulated. Interestingly, both Sox5 and Sox9 genes coordinately responded to changes in US stimulation and generally mirrored the response of collagen type II transcript to changes in US stimulation. RT-PCR analysis revealed that US stimulation increased the gene expression of cell-surface integrins α5 and ß1. The expression of integrins α2 was downregulated by US treatment, suggesting that multiple integrin subunits may be involved in the regulation of chondrocytic function in response to US stimuli. The enhancement in the abundance of the mRNA transcripts upon US stimulation was observed to correlate with the protein expression of collagen type I, collagen type II, and integrins α5 and ß1. In conclusion, the US stimulation regimen employed was shown to modulate the proliferative capacity, biosynthetic activity and integrin mRNA expression of articular chondrocytes maintained in 3D matrices.

Antibodies to plant-produced Plasmodium falciparum sexual stage protein Pfs25 exhibit transmission blocking activity.

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.

Green algae Chlamydomonas reinhardtii possess endogenous sialylated N-glycans.

Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii, a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry analysis of N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii and demonstrated that C. reinhardtii algae possess glycoproteins with mammalian-like sialylated N-linked oligosaccharides. These findings suggest that C. reinhardtii may be an attractive system for expression of target proteins.

Automated two-column purification of iminobiotin and BrdU-labeled PCR products for rapid cloning: application to genes synthesized by polymerase chain assembly.

Polymerase chain assembly (PCA) is a powerful tool for basic biological research and biotechnology applications. During the last several years, major advances have been made in de novo gene synthesis. However, there is still a need for fast and reproducible methods to automatically purify the synthesized genes. Upon completion of PCA, the subsequent PCR-amplified product mixture still contains undesired shorter DNA fragments that hinder cloning efforts. To avoid tedious gel purification, an automated two-column purification has been developed and used in conjunction with rapid PCA. The system enables fast synthesis and isolation of the full-length DNA of interest, important for facile cloning of desired DNA fragments. During the PCR amplification step, forward and reverse primers tagged with iminobiotin and bromodeoxyuridine labels, respectively, were used. The automated purification was then performed on the PCR mixture using two affinity/immunocapture columns in series to isolate only the desired full-length product. The procedure has been applied to the pUC19 beta-lactamase gene (929 bp). Follow-up PCR of the purified product, cloning, and sequencing demonstrated the technique's effectiveness in obtaining the pure full-length gene. The purification has also been performed on other synthesized genes, indicating its utility as a general approach.

Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler.

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653bp) and pUC19 beta-lactamase (929bp) in under 20min. However, rapid integrated PCA-PCR was found to be problematic when attempted with the TM-1 gene (1509bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers.

Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein.

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His(6)-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro.

Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes.

The assembly of synthetic oligonucleotides into genes and genomes is an important methodology. Several methodologies for such synthesis have been developed, but they have two drawbacks: (1) the processes are slow and (2) the error frequencies are high (typically 1-3 errors/kb of DNA). Thermal damage is a major contributor to biosynthetic errors. In this paper, we elucidate the advantages of rapid gene synthesis by polymerase chain assembly (PCA) when used in combination with smart error control strategies. We used a high-speed thermocycler (PCRJet) to effectively minimize thermal damage and to perform rapid assembly of synthetic oligonucleotides to construct two different genes: endothelial protein C receptor (EPCR) and endothelial cell thrombin receptor, thrombomodulin (TM). First, the intact EPCR gene (EPCR-1, 612 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively. Next, for direct error comparison, another longer gene, the 1548 bp TM gene was constructed from 87 oligonucleotides by both rapid and conventional PCA. The fidelity and accuracy of the synthetic genes generated in this manner were confirmed by sequencing. The combined steps of PCA and DNA amplification are completed in about 10 and 22 min for EPCR-1, 2 and TM genes, respectively with comparable low errors in the DNA sequence. Furthermore, we subcloned synthetic TM, EPCR-1, EPCR-2 and native EPCR-1 (amplified from cDNA) into a Pichia pastoris expression vector to evaluate the expression ability, and to compare them with the native gene. Here, we illustrate that the synthetic genes, assembled by rapid PCA, successfully directed the expression of functional proteins. And, importantly, the synthetic and the native genes expressed proteins with the same efficiency.

The two divergent PEP-carboxylase catalytic subunits in the green microalga Chlamydomonas reinhardtii respond reversibly to inorganic-N supply and co-exist in the high-molecular-mass, hetero-oligomeric Class-2 PEPC complex.

Our recent molecular studies revealed two divergent PEP-carboxylase (PEPC [Ppc]) encoding genes in the green microalga Chlamydomonas reinhardtii, CrPpc1 and CrPpc2, which are coordinately responsive to changes in inorganic-N and -C supply at the transcript level [Mamedov, T.G., Moellering, E.R. and Chollet, R. (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga C. reinhardtii, Plant J. 42, 832-843]. Here, we report the distribution of these two encoded catalytic subunits in the minor Class-1 and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, hetero-oligomeric complex containing both CrPpc1 (p109) and CrPpc2 (p131) polypeptides. The Class-1 enzyme, however, is a typical PEPC homotetramer comprised solely of p109. We also document that the amount of both CrPpc1/2 catalytic subunits is up-/down-regulated by varying levels of NH(4)(+) supplied to the culture medium.