Kulvinder Singh Saini

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.

The structure, metabolism and function of the carcinoembryonic antigen gene family

II. CEA structure and heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 A. Structure of the polypeptide backbone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 B. Structure of the oligosaccharide chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 C. The domain model for carcinoembryonic antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 D. Relationship to the immunoglobulin supergene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Molecular analysis of PTEN and MXI1 in primary bladder carcinoma.

Loss of heterozygosity (LOH) on 10q is associated with late‐stage events in urothelial neoplastic progression. The tumor suppressor gene PTEN, which is mutated or homozygously deleted in numerous cancers, maps to a region of 10q within the reported region of minimal loss in bladder tumors. In two recent studies alterations in the PTEN gene occur at a low frequency in bladder tumors displaying 10q LOH. We have screened 35 late‐stage bladder tumors for mutations in PTEN and MXI1, both genes mapping to chromosome 10q. Using single‐strand conformation polymorphism analysis, we identified 6 tumors harboring mutations in PTEN and 2 additional tumors displaying homozygous deletion at this locus. No MXI1 mutations were identified within the same tumor panel. Of 16 bladder tumor cell lines analyzed, 2 showed homozygous deletion of PTEN and 3 harbored point mutations resulting in an amino acid change. Two cell lines harbored missense mutations in MXI1. We report a significantly higher frequency of PTEN alterations in bladder carcinoma (23%) than was previously recorded, with no accompanying mutations in the MXI1 gene. Int. J. Cancer 88:620–625, 2000.

RNA interference therapeutics for cancer: Challenges and opportunities (Review)

RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism in animals and plants, which is mediated by double-stranded RNA (dsRNA). There has recently been an increasing interest in harnessing the gene silencing activity of dsRNA to develop novel drugs for the treatment of various diseases, such as cancer, neurological disorders, age-related macular degeneration and viral infections. Small interfering RNA (siRNA)-based drugs have distinct advantages over conventional small molecule or protein-based drugs, including high specificity, higher potency and reduced toxicity. However, there are several technical obstacles to overcome before siRNA-based drugs reach the clinic. Delivery of siRNA to the target tissues and stability in the serum remain a major challenge and are the main focus of current research and development efforts. This review focused primarily on the progress made in developing RNAi as therapeutics for cancer and the challenges associated with its clinical development. Use of ligands recognizing cell-specific receptors to achieve tumor-specific delivery of siRNA, methods for enhanced siRNA delivery, improving the bioavailability and pharmacokinetic properties of siRNA and reducing the off-target effects and non-specific gene silencing are discussed in the light of current evidence.

Cell factories for insulin production

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, cost-effectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

Molecular events regulating messenger RNA stability in eukaryotes.

The regulation of mRNA turnover plays a major role in the overall control of gene expression. Transcriptional control of eukaryotic gene regulation by external and/or internal stimuli has received considerable attention and the purpose of this review is to highlight recent work elucidating the mechanisms underlying the steady-state levels of mRNAs in the cytoplasm. Protection of mRNA from the action of nucleases as it passes from the nucleus to the ribosomes for translation is achieved, at least in part, by its union with mRNA binding proteins and the presence of poly(A) tail. The half-life of a message represents a balance between the transcriptional activity and intracellular degradative processes. These properties can be modulated by the presence of specific nucleotide sequences in a mRNA along with cis- and trans-acting elements and accompanied by post-translational feed back mechanisms. Presently, various regulatory mechanisms involved in the mRNA decay process are ill-defined. The work described here illustrates the complexity of this emerging field of study and outlines its contribution to our understanding of gene regulation in eukaryotes.

Mode of Action of Ranbezolid against Staphylococci and Structural Modeling Studies of Its Interaction with Ribosomes

ABSTRACT Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively. The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity against the target pathogen.

Cancer stem cells: a challenging paradigm for designing targeted drug therapies

Despite earlier controversies about their role and existence within tumors, cancer stem cells (CSCs) are now emerging as a plausible target for new drug discovery. Research and development (R&D) efforts are being directed against key gene(s) driving initiation, growth, and metastatic pathways in CSCs and the tumor microenvironment (TME). However, the niche signals that enable these pluripotent CSCs to evade radio- and chemotherapy, and to travel to secondary tissues remain enigmatic. Small-molecule drugs, biologics, miRNA, RNA interference (RNAi), and vaccines, among others, are under active investigation. Here, we examine the feasibility of leveraging current knowhow of the molecular biology of CSCs and their cellular milieu to design futuristic, targeted drugs with potentially lower toxicity that can override the multiple drug-resistance issues currently observed with existing therapeutics.

Dictyostelium discoideum—a promising expression system for the production of eukaryotic proteins

In general, four different expression systems, namely, bacterial, yeast, baculovirus, and mammalian, are widely used for the overproduction of biochemical enzymes and therapeutic proteins. Clearly, bacterial expression systems offer ease of maneuver ability with respect to large‐scale production of recom binant proteins, while, a baculovirus expression system ensures proper protein modifications, processing, and refolding of complex proteins. Despite these advan tages, mammalian cells remain the preferred host for many eukaryotic proteins of pharmaceutical impor tance, particularly, those requiring post‐translational modifications. Recently, the single‐celled slime mold, Dictyostelium discoideum (Dd), has emerged as a prom ising eukaryotic host for the expression of a variety of heterologous recombinant eukaryotic proteins. This organism possesses the complex cellular machinery required for orchestrating post‐translational modifica tions similar to the one observed in higher eukaryotes. This review summarizes the advantages and disadvantages of Dictyostelium as an alternate system compared to other well‐established expression systems. The key lessons learned from the expression of human recombinant pro teins in this system are reviewed. Also, the strengths, weaknesses, and challenges associated with industrial scale production of proteins in Dd expression system are discussed.— Arya, R., Bhattacharya, A., Saini, K. S. Dictyostelium discoideum—a promising expression system for the production of eukaryotic proteins. FASEB J. 22, 4055–4066 (2008)