Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.     

An Improved Bioprocess for Extracellular l-Leucine Amino Peptidase Production Using Streptomyces gedanensis

A bioprocess was developed for the production of L-leucine aminopeptidase under solid-state fermentation (SSF) by cultivating Streptomyces gedanensis in an inert support impregnated with a minimal medium. Response surface methodology of Box Behnken design was used to derive the optimum level of significant factors (3 ml inoculum (1.2 × 10(9) CFU/ml); 0.275% w/v (NH(4))(2)SO(4); 0.275% w/v MgSO(4)·7H(2)O and 0.55% w/v Tryptone) for maximum LAP production (489 IU/g PUF) as compared to the initial level of 176.3 ± 0.02 IU/g PUF. The high level of extracellular aminopeptidase yield achieved in this work showed the technical feasibility of LAP production under SSF using inert support and is the first report of this kind. The ability of Streptomyces amino peptidase to release particular N-terminal amino acids made them interesting for controlling the degree of hydrolysis and flavor development for a wide range of substrates in food like industries.     

Anchorage of GFP fusion on the cell surface of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Here we report the cell surface display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion by employing the N- and C-terminal domains of ice nucleation protein (INPNC) as an anchoring motif. An E. coli–Pseudomonas shuttle vector, pNOG33, coding for INPNC–OPH–GFP was constructed for targeting the fusion onto the cell surface of p-nitrophenol (PNP)-degrading P. putida JS444. The surface localization of INPNC–OPH–GFP was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH–GFP was demonstrated by OPH assays and fluorescence measurements. Surface display of macromolecular OPH–GFP fusion (63 kDa) neither inhibited cell growth nor affected cell viability. These results suggest that INP is an useful tool for the presentation of heterologous proteins on cell surfaces of indigenous microbes. The engineered P. putida JS444 degraded organophosphates (OPs) as well as PNP rapidly and could be easily monitored by fluorescence. Parathion (100 mg kg⁻¹) could be degraded completely within 15 days in soil inoculated with the engineered strain. These merits make this engineered strain an ideal biocatalyst for in situ bioremediation of OP-contaminated soil.     

Protein acyltransferase function of purified calreticulin: the exclusive role of P-domain in mediating protein acylation utilizing acyloxycoumarins and acetyl CoA as the acyl group donors

The distinct biochemical function of endoplasmic reticulum (ER) protein Calreticulin (CR) catalyzing the transfer of acyl group from acyloxycoumarin to a receptor protein was termed calreticulin transacylase (CRTAase). The present study, unlike the previous reports of others utilizing CR-deficient cells alone, dealt with the recombinant CR domains of Heamonchus contortus (rhCRTAase) in order to examine their CRTAase activity. P-domain of rhCR unlike N- and C-domains was found to be endowed with CRTAase function. We have also observed for the first time acetyl CoA, as a substrate for rhCRTAase/P-domain mediated acetylation of recombinant Schistosoma japonicum glutathione S-transferase (rGST). rhCRTAase/P-domain were also found to undergo autoacylation by acyloxycoumarins. Also, the isolated autoacylated rhCRTAase/P-domain in non-denatured form alone exhibited the ability to transfer acyl group to rGST indicating the stable intermediate nature of acylated CR. P-domain catalyzed acetylation of rGST by 7,8-Diacetoxy-4-methylcoumarin or acetyl CoA resulted in the modification of several lysine residues in common was evidenced by LC-MS/MS analysis. The putative site of the binding of acyloxycoumarins with CR was predicted by computational blind docking studies. The results showed the involvement of two lysine residues Lys-173 and Lys-174 present in P-domain for binding acyloxycoumarins and acetyl CoA thus highlighting that the active site for the CRTAase activity would reside in the P-domain of CR. Certain ER proteins are known to undergo acetylation under the physiological conditions involving acetyl CoA. These results demonstrating CRTAase mediated protein acetylation by acetyl CoA may hint at CR as the possible protein acetyltransferase of the ER lumen.     

Radical scavenging and radiomodulatory effects of Psoralea corylifolia Linn. substantiated by in vitro assays and EPR spectroscopy

The present study is the first report of the radiomodulatory effects of Psoralea corylifolia Linn. The extract (IBG-RA-26) prepared from P. corylifolia was chemically analysed by HPLC, LC-MS/MS and NMR. The total polyphenolic content of IBG-RA-26 was 0.287 mg/ml of quercetin equivalents. IBG-RA-26 exhibited a dose-dependent increase in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. It exhibited comparable (> 50%) site-specific and non-site-specific hydroxyl radical scavenging activity in higher concentration ranges (500-1000 microg/ml), while at lower concentrations (5-50 microg/ml) it exhibited significantly (p < 0.05) higher non-site-specific scavenging ability compared to site-specific activity. Nitric oxide scavenging activity of IBG-RA-26 (5-1000 microg/ml) increased in a concentration-dependent manner, while maximum superoxide ion scavenging ability (79%) was observed at 50 microg/ml. The electron donation potential of IBG-RA-26 was found to be higher than that of ascorbic acid at lower concentrations (up to 5 microg/ml). Analysis of the ability of IBG-RA-26 to protect membranes against gamma-radiation, utilizing an artificial membrane system (liposome), revealed a significant (p < 0.05) decrease in the formation of malondialdehyde (MDA) as a function of the concentration of IBG-RA-26. Radiation-induced lysis of human erythrocytes was monitored and efficacy of IBG-RA-26 was tested in the concentration range 25-1000 microg/ml, with significant protective efficacy observed in the range 25-50 microg/ml. IBG-RA-26 rendered significant (p < 0.05) protection against radiation (0.25 kGy)-induced DNA damage. EPR spectroscopy was used to investigate the DPPH radical scavenging capacity of IBG-RA-26. IBG-RA-26 exhibited a good DPPH radical scavenging capacity in a concentration-dependent manner. By direct EPR spectroscopy we have also demonstrated the possible formation of free radical species in a solution of IBG-RA-26. The wide spectrum of radioprotective and antioxidant properties exhibited by IBG-RA-26 indicate that P. corylifolia has potential as a radiomodulatory agent.     

Identification of Potential Peptide Inhibitors of ACE-2 Target of SARS-CoV-2 from Buckwheat & Quinoa

International Journal of Peptide Research and Therapeutics - It is well established fact that peptides from various foods offer human health benefits displaying diverse functionalities. Millets...     

Platelet-rich plasma vs bone marrow aspirate concentrate: An overview of mechanisms of action and orthobiologic synergistic effects

The use of orthobiologics as a novel therapy for the treatment of numerous musculoskeletal disorders has increased considerably over the past decade.Currently,there are multiple alternatives available as suitable treatments;however,the use of autologous blood-derived products such as platelet-rich plasma(PRP),bone marrow aspirate(BMA)and BMA concentrate(BMAC),specifically,is expanding.Although many investigations attempted to demonstrate the effectiveness of these therapies,even with positive results,the literature lacks standardized protocols and overall accuracy in study designs,which leads to variance and difficulty in reproducibility of protocols.The efficacy of PRP for the treatment of cartilage,bone and muscle tissues is well known.Although BMAC has generated optimistic results for the same purposes,its applicability in clinical trials is still relatively recent when compared to PRP.Both products demonstrate the potential to set forth reparative processes,each in their own distinct mechanism.The combination of these biological products has been previously proposed,yet little is known about their synergism.Evidence indicates that growth factor,cytokine,and chemokine profiles seen in both PRP and BMAC vary but are likely to work synergistically to enhance musculoskeletal healing.BMAC products seem to work well without PRP;however,the addition of PRP to BMAC has been shown to act as a rich and natural source of culture medium for stem cells located either peripherally or in the bone marrow itself.Nevertheless,additional variables associated with the use of BMAC and PRP in orthopedics must be further evaluated in order to consolidate the efficacy of this therapeutic strategy.     

Ocimum sanctum aqueous leaf extract provides protection against mercury induced toxicity in Swiss albino mice

HgCl2 (5.0 mg/kg body weight) induced toxicity led to significant elevation of lipid peroxidation (LPO) level but decline in the glutathione content in liver of Swiss albino mice. In serum of HgCl2 treated mice there was significant elevation in serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) activities but significant decline in the alkaline phosphatase activity. Animals treated with O. sanctum extract (10 mg/kg body weight, po) before and after mercury intoxication showed a significant decrease in LPO level, SGOT and SGPT activities and increase in serum alkaline phosphatase activity and glutathione (GSH) content. Ocimum treatment alone did not alter SGOT, SGPT and alkaline phosphatase activities but significantly enhanced reduced glutathione. The results suggest that oral administration of Ocimum extract provides protection against HgCl2 induced toxicity in Swiss albino mice.     

Unraveling biochemical properties of cockroach (Periplaneta americana) proteinases with a gel X-ray film contact print method

Eleven proteinase activity bands were detected in American cockroach (Periplaneta americana) gut. These were partially purified and characterized using a gel X-ray film contact print method. Cockroach gut proteinases (CGPs) show activity over a broad range of pH with maximum activity between pH 6 and 10, and optimal activity at 50-70 degrees C. CGPs were partially purified by preparative gel electrophoresis and analyzed using synthetic substrates and inhibitors. Four of the proteases exhibited chymotrypsin-like (C1 to C4) activity and seven trypsin-like (T1 to T7) activity. Accuracy of the gel X-ray film contact print method is confirmed by including bovine chymotrypsin in CGP analysis. Inhibition of CGPs with different plant proteinaceous proteinase inhibitors allowed identification of potential CGP inhibitors. Our results imply that presence of several CGP activity bands, and their stability and activity over a broad pH and temperature range might contribute to adaptation of P. americana to extreme environmental conditions and the polyphagous nature of the species.     

Studies on wound healing potential of red pigment isolated from marine Bacterium Vibrio sp.

Wounds are common clinical entities of life which may be subacute or acute. Wound healing is a complex biochemical process where the cell structures are restored to normalcy, which depend on cell proliferation and migration, basically fibroblast cell. The present investigation was undertaken to evaluate the healing efficacy of red pigment isolated from marine isolate Vibrio sps on experimental wounds in albino rats. The red pigment was applied topically, twice daily for 14 days. Treatment with framycetin ointment was used as reference control. The red pigment treated group showed faster reduction in wound area in comparison with control and framycetin ointment treated groups. In conclusion, red pigment possesses significant healing potential in wounds and has a positive influence on the different phases of wound repair.