Hamid Bashir

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.

Exosomes as Biomarker of Cancer

Rapid advances in medicine and biotechnology resulted in the development of non-invasive diagnostic and prognostic biomarkers enabling convenient and accurate detection. Exosomes has recently emerged as non-invasive biomarker for a number of diseases including cancer. Exosomes are the small endosome originated membranous vesicles secreted in a number of biological fluids such as serum, saliva, urine, ascites, cerebrospinal fluid, etc. Exosomes contain microRNA proteins and mRNA which can be used as disease specific biomarkers. Here we reviewed recent advancement in the field of exosomes as diagnostic biomarker for cancer along with a brief overview of their biogenesis, function and isolation.

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.

Developing a deeper insight into reproductive biomarkers

The development of biomarkers of reproductive medicine is still in its infancy because many black boxes are still present in reproductive medicine. Novel approaches to human infertility diagnostics and treatment must be developed because reproductive medicine has lagged behind in the implementation of biomarkers in clinical medicine. Despite the dearth of the available literature, the current rapid pace of publications suggests that this gap will soon be filled therefore; this review is a précis of the research that has been done so far and will provide a basis for the development of biomarkers in reproductive medicine.

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.