Sudhir Sahdev

Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies

Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein’s over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.

Host-pathogen interactions during apoptosis.

Host pathogen interaction results in a variety of responses, which include phagocytosis of the pathogen, release of cytokines, secretion of toxins, as well as production of reactive oxygen species (ROS). Recent studies have shown that many pathogens exert control on the processes that regulate apoptosis in the host. The induction of apoptosis upon infection results from a complex interaction of parasite proteins with cellular host proteins. Abrogation of host cell apoptosis is often beneficial for the pathogen and results in a successful host invasion. However, in some cases, it has been shown that induction of apoptosis in the infected cells significantly imparts protection to the host from the pathogen. There is a strong correlation between apoptosis and the host protein translation machinery: the pathogen makes all possible efforts to modify this process so as to inhibit cell suicide and ensure that it can survive and, in some cases, establish latent infection. This review discusses the significance of various pathways/steps during virus-mediated modulation of host cell apoptosis.

Antioxidants prevent UV-induced apoptosis by inhibiting mitochondrial cytochrome c release and caspase activation in Spodoptera frugiperda (Sf9) cells.

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV‐induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV‐induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf‐caspase‐1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced‐glutathione (GSH) on ultra violet B (UVB)‐induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf‐caspase‐1 activation was inhibited by pre‐treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.

Indian herb 'Sanjeevani' (Selaginella bryopteris) can promote growth and protect against heat shock and apoptotic activities of ultra violet and oxidative stress

Selaginella bryopteris is a lithophyte with remarkable ressurection capabilities. It is full of medicinal properties, hence also known as ‘Sanjeevani’ (one that infuses life). For lack of credible scientific evidence the plant is not in active use as a medicinal herb. We provide scientific evidence for whyS. bryopteris is known as ‘Sanjeevani’.The aqueous extract ofS. bryopteris possesses growth-promoting activity as well as protective action against stress-induced cell death in a number of experimental cell systems including mammalian cells. Treatment of the cells in culture with 10% aqueous extract enhanced cell growth by about 41% in Sf9 cells and 78% in mammalian cells. Pre-treatment of cells with the Selaginella extract (SE) (1-2x5%) protected against oxidative stress (H2O2)-induced cell death. The killing potential of ultra violet (UV) was also significantly reduced when the cells were pre-treated with SE for 1 h. Thermal radiation suppressed cell growth by about 50%. Pre-treatment of cells with SE for 1 h afforded complete protection against heat-induced growth suppression. SE may possess anti-stress and antioxidant activities that could be responsible for the observed effects. Chemical analysis shows that SE contains hexoses and proteins. Taken together,S. bryopteris extract may help in stress-induced complications including those due to heat shock.

Baculoviral p35 inhibits oxidant-induced activation of mitochondrial apoptotic pathway.

In this study we report that the baculovirus p35 anti-apoptotic protein prevents cell death by quenching free radicals at a very upstream step in the apoptotic pathway. Mitochondria of activated rat peritoneal macrophages as well as Spodoptera frugiperda (Sf9) insect cells, following treatment with oxidants, H(2)O(2)/UVB irradiation, release cytochrome c followed by activation of caspase-3. Transfection of macrophages/Sf9 cells with a construct carrying the p35 gene under the CMV/HSP promoters resulted in p35 expression and consequent arrest of oxidative stress-induced apoptosis. p35 expression also inhibited cytochrome c release from the mitochondria of oxidant-exposed cells and blocked caspase-3 activation.

Invitro culturedSpodoptera frugiperda insect cells: Model for oxidative stress-induced apoptosis

Cellular imbalance in the levels of antioxidants and reactive oxygen species (ROS) is directly associated with a number of pathological states and results in programmed cell death or apoptosis. We demonstrate the use ofin vitro culturedSpodoptera frugiperda (sf9) insect cells as a model to study oxidative stress induced programmed cell death. Apoptosis ofin vitro cultured sf9 cells was induced by the exogenous treatment of H2O2 to cells growing in culture. The AD50 (concentration of H2O2 inducing about 50% apoptotic response) varied with the duration of treatment, batch to batch variation of H2O2 and the physiological state of cells. At 24 h post-treatment with H2O2 AD50 was about 475 Μm. Apoptosis could also be induced byin situ generation of H2O2 by the inhibition of catalase activity upon hydroxylamine treatment. Hydroxylamine acted synergistically with H2O2 with an AD50 of 2.2 mM. DMSO, a free radical scavenger, inhibited H2O2-induced apoptosis thereby confirming the involvement of reactive oxygen species. Exposure of cells to UV radiation (312 nm) resulted in a dose-dependent induction of apoptosis. These results provide evidence on the novel use of insect cells as a model for oxidative stress-induced apoptosis.

Enhanced expression of recombinant proteins utilizing a modified baculovirus expression vector

The baculovirus expression vector system (BEVS) has been widely used for over-expressing eukaryotic proteins due to a close resemblance in post-translational modification, processing, and transportation properties of the expressed protein, to that of the mammalian cells. In comparison to the bacterial expression system, protein yield from BEVS is relatively low, resulting in higher cost of production. To improve the existing recombinant protein expression levels, baculovirus homologous region1 (hr1) was strategically integrated into the bacmid-based transfer vectors. Luciferase reporter, human Protein Kinase B-α (PKB-A), and N-terminal-modified CYP-1A2 genes were independently cloned in non-hr1 and hr1 constructs for generating respective bacmids and baculoviruses. These recombinant baculoviruses were utilized for comparing the expresion levels at varying multiplicity of infections (MOI) and time intervals in Spodoptera frugiperda (Sf21) or Trichoplusia ni (Tni) insect cell lines. Targeted insertion of hr1 upstream to CYP-1A2, PKB-A, and Luciferase genes, compared to the non-hr1 sets, led to 3-, 3.5-, and 4.5-fold increase in the resultant protein levels, respectively. Moreover, at equal protein concentration, the corresponding activity and inhibition characteristics of these high expression hr1 sets were comparable to that of the respective non-hr1 sets. Utilization of this modified baculovirus expression construct offers significant advantage of producing recombinant proteins in a cost-effective manner for various biotechnological and therapeutic applications.

Baculovirus P35 protein: an overview of its applications across multiple therapeutic and biotechnological arenas.

Baculovirus immediate early P35 protein is well known for its anti‐apoptotic as well as anti‐oxidant properties. Mechanism of action of P35 involves inhibition of a vast range of initiator to executioner class of caspases. In addition, P35s role in inhibiting oxidant‐induced mitochondrial damage, primarily in the apoptotic pathway, has also been extensively investigated. Elucidation of P35s functions during regulation of programmed cell death (PCD) has led to a renewed focus on exploiting this basic knowledge for clinical and other related applications. This review outlines specific biochemical and genetic pathways where P35 intervenes and regulates rate‐limiting steps in the apoptotic signaling cascade. Research efforts are underway to utilize P35 as an agent in regulating apoptosis and under certain circumstances, also explore the therapeutic potential of its anti‐oxidant features. One of the major outcomes of recent studies include significantly improved effectiveness of cytochrome P450 directed enzyme pro‐drug delivery tools when used in conjunction with P35, which may help in alleviating drug resistance in tumor cells and simultaneously prolonging the cytotoxic effects of anti‐cancer drugs. Moreover, applied research carried out recently in the fields of diabetes, ischemia‐induced neuronal cell death, experimental autoimmune encephalomyelitis (EAE), multiple sclerosis (MS), inflammatory arthritis, cardiovascular and ocular disorders illustrate P35s utilization across diverse therapeutic areas and will certainly make it an attractive biomolecule for the discovery research.

Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

Spodoptera frugiperda FKBP-46 is a consensus p53 motif binding protein

p53 protein, the central molecule of the apoptosis pathway, is mutated in 50% of the human cancers. Of late, p53 homologues have been identified from different invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Squid, and Clams. We report the identification of a p53‐like protein in Spodoptera frugiperda (Sf9) insect cells, which is activated during oxidative stress, caused by exposure to UV‐B or H2O2, and binds to p53 consensus DNA binding motifs as well as other p53 cognate motifs. Sf9 p53 motif‐binding protein is similar to murine and Drosophila p53 in terms of molecular size, which is around 50–60 kDa, as evident from UV cross‐linking, and displays DNA binding characteristics similar to both insect and vertebrate p53 as seen from electrophoretic mobility shift assays. The N‐terminal sequencing of the purified Sf9 p53 motif‐binding protein reveals extensive homology to the pro‐apoptotic FK‐506 binding protein (FKBP‐46), earlier identified in Sf9 cells as a factor which interacts with murine casein kinase. FKBP, an evolutionarily conserved protein of mammalian origin functions as a pro‐apoptotic factor. Identification of FKBP‐46 as a novel p53 motif‐binding protein in insect cells adds a new facet to our understanding of the mechanisms of apoptosis under oxidative stress in the absence of a typical p53 homologue. J. Cell. Biochem. 114: 899–907, 2013.