Nadeem Ahmed

Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN.

Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl‐β‐d‐thiogalactopyranoside (IPTG), a non‐metabolizable and expensive compound, as inducer. For economical and commercial‐scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single‐step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot‐scale production of recombinant proteins at low cost.

Optimization of conditions for high‐level expression and purification of human recombinant consensus interferon (rh‐cIFN) and its characterization

Recombinant human consensus interferon (rh‐cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high‐level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high‐level expression, shake‐flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l‐arginine were optimized. A one‐step chromatography method was developed for high‐yield purification of consensus IFN. rh‐cIFN was characterized by SDS‐PAGE, Western blot, and high‐performance liquid chromatography. Purified protein has a molecular weight of 19.5 kDa and specific activity was 2.0 × 108 as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100 mg/L of biologically active rh‐cIFN, which is highest ever reported according to available data.

Matrix-assisted refolding and purification of placenta-derived recombinant human interleukin-6 produced in Escherichia coli

Biological activity of human interleukin‐6 (IL‐6) is associated with a vast number of diseases such as rheumatoid arthritis, sepsis, and severe inflammatory diseases. In this study, human IL‐6 cDNA was isolated from a cDNA library that was constructed with mRNA derived from human placental tissues. Subsequently, the complete human IL‐6 cDNA was cloned and expressed in BL21DE3 cells. The recombinant human IL‐6 (rhIL‐6) protein was expressed in a form of an insoluble inclusion body. Inclusion bodies were solubilized under denaturing conditions and purified by immobilized metal affinity chromatography with gradual on‐column refolding by the gradient elution method (from 6 to 0 M urea). The protein was purified to apparent homogeneity of about 99% with a yield of 50 mg/L. The purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), size exclusion high‐performance liquid chromatography, and Western blotting analysis. The bioactivity was assessed by proliferation assay of TF‐1 cells in a dose‐dependent manner. The present study confirms the expression of the placenta‐derived IL‐6 gene in a prokaryotic expression system and matrix‐assisted on‐column refolding and purification of rhIL‐6 by immobilized metal affinity chromatography.

Prediction of Potato Late Blight Disease Based upon Environmental Factors in Faisalabad, Pakistan

Potato late blight (PLB) caused by Phytophthora infestans (Mont.) de Bary, is an important and serious threat to successful potato production in the world. It spreads through seed and soil residual material. In Pakistan, PLB disease can induce 100% yield losses under epidemic condition. Due to lack of resistance in indigenous potato germplasm, disease is managed through fungicides by the growers of Pakistan. Excessive use of fungicides causes resistance in the pathogen and creates fatalistic effect on the environment. Disease predictive model under such situation may be effective tool to predict early onset of disease. A disease predictive model was developed on two years data of PLB disease severity and epidemiological factors using stepwise regression analysis. Model explained upto 80% disease variability. Maximum and Minimum temperature, relative humidity, rainfall and wind speed appeared to be most significant factors in the PLB disease development. The environmental conditions conducive for the development of PLB disease were characterized. Maximum and minimum temperatures in the range of 16-20°C and 1-6°C were found favourable for potato blight disease. Similarly, relative humidity, rainfall and wind speed in the range of 63-71%, 1.5-3.75 mm and 1-5.5 Km/h, respectively, were conducive for PLB disease which are helpful in disease development.

Epidemiology, Pathogenesis, and Control of a Tick-Borne Disease- Kyasanur Forest Disease: Current Status and Future Directions

In South Asia, Haemaphysalis spinigera tick transmits Kyasanur Forest Disease Virus (KFDV), a flavivirus that causes severe hemorrhagic fever with neurological manifestations such as mental disturbances, severe headache, tremors, and vision deficits in infected human beings with a fatality rate of 3–10%. The disease was first reported in March 1957 from Kyasanur forest of Karnataka (India) from sick and dying monkeys. Since then, between 400 and 500 humans cases per year have been recorded; monkeys and small mammals are common hosts of this virus. KFDV can cause epizootics with high fatality in primates and is a level-4 virus according to the international biosafety rules. The density of tick vectors in a given year correlates with the incidence of human disease. The virus is a positive strand RNA virus and its genome was discovered to code for one polyprotein that is cleaved post-translationally into 3 structural proteins (Capsid protein, Envelope Glycoprotein M and Envelope Glycoprotein E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). KFDV has a high degree of sequence homology with most members of the TBEV serocomplex. Alkhurma virus is a KFDV variant sharing a sequence similarity of 97%. KFDV is classified as a NIAID Category C priority pathogen due to its extreme pathogenicity and lack of US FDA approved vaccines and therapeutics; also, the infectious dose is currently unknown for KFD. In India, formalin-inactivated KFDV vaccine produced in chick embryo fibroblast is being used. Nevertheless, further efforts are required to enhance its long-term efficacy. KFDV remains an understudied virus and there remains a lack of insight into its pathogenesis; moreover, specific treatment to the disease is not available to date. Environmental and climatic factors involved in disseminating Kyasanur Forest Disease are required to be fully explored. There should be a mapping of endemic areas and cross-border veterinary surveillance needs to be developed in high-risk regions. The involvement of both animal and health sector is pivotal for circumscribing the spread of this disease to new areas.

One step purification of biological active human interleukin-2 protein produced in yeast ( Pichia Pastoris )

Pharmacological importance of recombinant human interleukin-2 protein has increased the demand to establish effective, reliable and cost effective chromatography method for its production and purification on large scale. One step mimetic ligand affinity chromatography method for purification of mutated human recombinant interleukin-2 (mrhIL-2), from Pichia Pastoris is described with higher yield then reported before. The mrhIL-2 was expressed extracellularly under methanol inducible AOX1 promoter of P. pastoris. Extracellular expression of mrhIL-2 in the culture supernatant was ~210 mg/L. Cell free culture supernatant containing mrhIL-2 protein was concentrated and buffer exchanged by diafiltration by tangential flow filtration system. Different mimetic legends column from sigma were screened for efficient binding of mrhIL-2 in culture supernatant. Maximum binding was observed with Mimetic Blue SA P6XL and least with Mimetic Green 1 A6XL. One step Dye ligand affinity chromatography method was developed by using Mimetic Blue SA P6XL ligand for high level purification of mutant of interleukin-2. Final yield of purified protein was 115 mg/L and purity of 97%. The interleukin-2 protein prepared by this protocol was found to be monomeric based on SDS-PAGE, western blot and HPLC analysis. Purified protein was biological active as checked by cell proliferation assay. Key words: Interleukin-2, Pichia pastoris, Dye ligand affinity chromatography.

Glycosylation of Recombinant Anticancer Therapeutics in Different Expression Systems with Emerging Technologies

Glycosylation, a posttranslational modification, has a major role in recombinant anticancer therapeutic proteins, as most of the approved recombinant therapeutics are glycoproteins. The constant amino acid sequence of therapeutics determines the enzymatic activity, while the presence of glycans influences their pharmacokinetics, solubility, distribution, serum half-life, effector function, and binding to receptors. Glycoproteins expressed in different expression systems acquire their own oligosaccharides, which increases the protein diversity. The heterogeneity of glycans creates hurdles in downstream processing, ultimately leading to variable anticancer therapeutic efficacy. Therefore, glycoproteins require an appropriate expression system to obtain structurally and functionally identical glycans, as in humans. In many expression systems, the N-glycosylation pathway remains conserved in the endoplasmic reticulum, but divergence is observed when the protein enters the Golgi complex. Hence, in recent decades, numerous approaches have been adopted to engineer the Golgis N-glycosylation pathway to attain human-like glycans. Several researchers have tried to engineer the N-glycosylation pathway of expression systems. In this review, we examine the glycosylation pattern in various expression systems, along with emerging technologies for glycosylation engineering of anticancer therapeutic drugs. Cancer Res; 78(11); 2787-98. ©2018 AACR.

Enhancing recombinant interleukin‐6 production yield by fermentation optimization, two‐step denaturing, and one‐step purification

Interleukin‐6 a pleiotropic cytokine involved in a wide range of biological activities. So the large‐scale production of biologically active recombinant human interleukin‐6 is important for its structural and functional studies. Here, we report an optimized method for shake flask fermentation and a simplified high‐yield purification procedure for the recombinant interleukin‐6. This high‐yield expression method not only involves the optimization of the fermentation condition but also the single step purification method as well as a two‐step denaturing and one‐step refolding process. This approach replaces the more conventional procedure of protein solubilization and refolding. Through applying these strategies, the final cell density and overall product yield of the recombinant human interleukin‐6 were obtained as 20.4 g as cell biomass and 150 mg as purified active protein from the I‐L of the culture. The purified protein was characterized by HPLC and SDS–PAGE. The results of the current work demonstrate that the described method may be used to develop the process for industrial‐scale production of the biologically active recombinant interleukin‐6 protein.

Determination of mosquito Larvicidal potential of Bacillus thuringiensis Cry11Ba fusion protein through molecular docking

Mosquitoes spread deadly infections around the world. Since decades Bacillus thuringiensis (Bt) δ-endotoxins have been used successfully as a biopesticide for controlling mosquito larvae. However, over a few years, mosquito larvae have evolved tolerance against Bt δ-endotoxins, rendering them ineffective for mosquito control. Such a problem entails the development of improved toxins with enhanced toxicity, affinity towards a wide range of mosquito receptors and ability to overcome or delay the resistance buildup. In this study, using in silico tools, we aimed to design a fusion protein by fusing active region of Bt subsp. jegathesan Cry11Ba protein with Aedes aegypti TMOF (trypsin modulating oostatic factor). Using computational study, the fusion protein was validated and its mosquitocidal potential was determined through molecular docking against cadherin and aminopeptidase N midgut receptors of Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Molecular docking revealed that from Cry11Ba-TMOF fusion protein, domain II amino acids of Cry11Ba protein showed hydrogen bond interactions with cadherin and aminopeptidase N receptors of the targeted mosquitoes. These results conclude that Cry11Ba-TMOF fusion protein has a strong affinity for the receptors of Ae.aegypti, An.gambiae and Cx.quinquefasciatus. Thus the designed fusion protein can be used as a potent mosquitocidal agent for the control of targeted mosquitoes.

Evaluating the auto induction expression system and one step purification for high level expression and purification of gallbladder derived rhIL1Ra

Recent advancement in fermentation technologies resulted in the increased yields of recombinant proteins of biopharmaceutical and medicinal importance. Consequently, there is an important task to develop simple and easily scalable methods that can facilitate the production of high‐quality recombinant protein. Most of the recent reports described the expression of recombinant human IL‐1 receptor antagonist (rhIL‐1Ra) in Escherichia coli using isopropyl‐β‐d‐thiogalacto pyranoside (IPTG), a nonmetabolizable and expensive compound, as an expression inducer. In this study, we describe the expression and one‐step purification of gallbladder‐derived rhIL‐1Ra by autoinduction in E. coli. This method includes special media that automatically induce the target protein expression from T7 promoter and allow the production of the target protein in high yield than the conventional IPTG induction method. In addition to fermentation process improvements, one‐step purification strategy is essential to make the process economical. We developed a single‐step cation exchange chromatography and obtained 300 mg/L of rhIL‐1Ra with 98% purity. Purified protein was characterized by SDS‐PAGE and Ion exchange HPLC (IEX‐HPLC). The described method can be used to scale up the production of rhIL‐1Ra and other recombinant proteins.