Bioprocess Development for Recombinant Therapeutic Protein Osteopontin from Escherichia Coli

Osteopontin (OPN) is a multifunctional protein presented in various organs and body fluids in human body. OPN serves important roles in bone remolding, wound healing, and other biomineralization related physiological activities, which gives it great potential health benefits. Clinical statistics and laboratory researches also indicate that OPN can be a suitable diagnostic and prognostic marker for cancer progression. Its role in cell signaling remains one of the hot topics in biomedicine research. However, there was no efficient and scalable process for OPN purification from recombinant sources reported, which prevents OPN unleashing its full potential in health industry. Low expression level, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure are some of the challenges that faced purification development of OPN from Escherichia coli (E. coli) lysates. Finding an adequate capture step with sufficient specificity and binding capacity that could be paired with an orthogonal purification step was a vitally important objective for this project. In recent years, mixed-modal chromatography (MMC) resins have opened up new possibilities for protein purification. The combination of high-throughput screening (HTS) platforms and design of experiment (DOE) principles could accelerate process development exponentially. In this research, seven chromatography resins including traditional ion exchange resin, hydrophobic interaction resin and 5 mixed-modal resins were screened for OPN purification. Plate based HTS was applied and quickly revealed comprehensive information about the affinity between the ligands and the target protein, specificity of the adsorption, and the binding capacity of the resins. Based on HTS results, the bind/elute processes for all candidate resins were designed, tested and optimized with small scale batch adsorption experiments. The purification factors and OPN recoveries achieved by batch adsorption experiments were verified on packed-bed columns operated by ÄKTA system and analyzed statistically. In terms of purification factor and yield, HEA HyperCel and Capto Q performed significantly better than the rest of the candidate resins. Two 2-step purification processes assembled with these two resins elevated OPN purity from 2% from the lysate to over 95% purity with greater than 40% yield

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a "plant-like" algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN

Bioprocess Development for Recombinant Therapeutic Protein Osteopontin from Escherichia Coli

Osteopontin (OPN) is a multifunctional protein presented in various organs and body fluids in human body. OPN serves important roles in bone remolding, wound healing, and other biomineralization related physiological activities, which gives it great potential health benefits. Clinical statistics and laboratory researches also indicate that OPN can be a suitable diagnostic and prognostic marker for cancer progression. Its role in cell signaling remains one of the hot topics in biomedicine research. However, there was no efficient and scalable process for OPN purification from recombinant sources reported, which prevents OPN unleashing its full potential in health industry. Low expression level, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure are some of the challenges that faced purification development of OPN from Escherichia coli (E. coli) lysates. Finding an adequate capture step with sufficient specificity and binding capacity that could be paired with an orthogonal purification step was a vitally important objective for this project. In recent years, mixed-modal chromatography (MMC) resins have opened up new possibilities for protein purification. The combination of high-throughput screening (HTS) platforms and design of experiment (DOE) principles could accelerate process development exponentially. In this research, seven chromatography resins including traditional ion exchange resin, hydrophobic interaction resin and 5 mixed-modal resins were screened for OPN purification. Plate based HTS was applied and quickly revealed comprehensive information about the affinity between the ligands and the target protein, specificity of the adsorption, and the binding capacity of the resins. Based on HTS results, the bind/elute processes for all candidate resins were designed, tested and optimized with small scale batch adsorption experiments. The purification factors and OPN recoveries achieved by batch adsorption experiments were verified on packed-bed columns operated by ÄKTA system and analyzed statistically. In terms of purification factor and yield, HEA HyperCel and Capto Q performed significantly better than the rest of the candidate resins. Two 2-step purification processes assembled with these two resins elevated OPN purity from 2% from the lysate to over 95% purity with greater than 40% yield

Passive Detection of Ship-Radiated Acoustic Signal Using Coherent Integration of Cross-Power Spectrum with Doppler and Time Delay Compensations

Passive sonar is widely used for target detection, identification and classification based on the target radiated acoustic signal. Under the influence of Doppler, generated by relative motion between the moving target and the sonar array, the received ship-radiated acoustic signals are non-stationary and time-varying, which has a negative effect on target detection and other fields. In order to reduce the influence of Doppler and improve the performance of target detection, a coherent integration method based on cross-power spectrum is proposed in this paper. It can be concluded that the frequency shift and phase change in the cross-power spectrum obtained by each pair of data segments can be corrected with the compensations of time scale (Doppler) factor and time delay. Moreover, the time scale factor and time delay can be estimated from the amplitude and phase of the original cross-power spectrum, respectively. Therefore, coherent integration can be implemented with the compensated cross-power spectra. Simulation and experimental data processing results show that the proposed method can provide sufficient processing gains and effectively extract the discrete spectra for the detection of moving targets

Potential Application of Alternate Tillage (AT) in a Rice–Wheat Rotation System—Based on Soil Physical Properties, Wheat Growth and Yield

Alternate tillage (AT) has the potential to reduce inputs and improve soil quality and crop yield, but there has been no research on the effect of AT on soil and wheat in a rice–wheat rotation system. In this study, field experiments were conducted to examine the effects of four tillage management methods (conventional tilling (CT) in each crop (RCT–WCT), no tilling (NT) in rice and conventional tilling in wheat (RNT–WCT, AT1), conventional tilling in rice and no tilling in wheat (RCT–WNT, AT2), and no tilling in each crop (RNT–WNT)) on the physical properties of soil, wheat growth, and yield. At the 0–5 cm soil layer, CT in the wheat season increased bulk density (BD) and decreased total properties, but it decreased BD at the 5–40 cm soil layer, and the effect of RCT–WCT was significantly greater than that of RNT–WCT. CT in the wheat season increased the root activity, root dry weight, net photosynthetic rate, leaf area index, antioxidant enzyme activities, and yield, and there was no significant effect between RCT–WCT and RNT–WCT. RNT-WCT has the potential to reduce inputs and maintain wheat yields

Impact of Tillage and Straw Treatment Methods on Rice Growth and Yields in a Rice–Ratoon Rice Cropping System

The rice–ratoon rice cropping system has the advantages of saving labor and imparting economic benefits. Optimizing tillage and straw management is beneficial for improving ratoon rice growth and yield. In this study, field experiments were conducted to examine the effects of four tillage and straw managements on the growth and yield of a rice–ratoon rice cropping system in central China in 2020 to 2021. The managements included no-till with main-season and ratoon-season rice residues retained on the soil’s surface (NT+S), plow tillage with residue retention (CT+S), no-till with residues removed (NT-S), and plow tillage with residues removed (CT-S). Compared to NT, CT significantly increased yield by 33.70% and 29.12% in the main and ratoon seasons, respectively. Compared to straw removal, straw returning significantly increased yield by 13.37% and 27.29% in the main and ratoon seasons, respectively. In general, both CT and straw returning improved root function (root activity and root dry weight) and photosynthetic capacity (leaf area index, net photosynthetic rate, and leaf chlorophyll content). CT combined with straw returning was able to achieve the highest annual rice yield

Impact of Tillage and Straw Treatment Methods on Rice Growth and Yields in a Rice–Ratoon Rice Cropping System

The rice–ratoon rice cropping system has the advantages of saving labor and imparting economic benefits. Optimizing tillage and straw management is beneficial for improving ratoon rice growth and yield. In this study, field experiments were conducted to examine the effects of four tillage and straw managements on the growth and yield of a rice–ratoon rice cropping system in central China in 2020 to 2021. The managements included no-till with main-season and ratoon-season rice residues retained on the soil’s surface (NT+S), plow tillage with residue retention (CT+S), no-till with residues removed (NT-S), and plow tillage with residues removed (CT-S). Compared to NT, CT significantly increased yield by 33.70% and 29.12% in the main and ratoon seasons, respectively. Compared to straw removal, straw returning significantly increased yield by 13.37% and 27.29% in the main and ratoon seasons, respectively. In general, both CT and straw returning improved root function (root activity and root dry weight) and photosynthetic capacity (leaf area index, net photosynthetic rate, and leaf chlorophyll content). CT combined with straw returning was able to achieve the highest annual rice yield

Potential Application of Alternate Tillage (AT) in a Rice–Wheat Rotation System—Based on Soil Physical Properties, Wheat Growth and Yield

Alternate tillage (AT) has the potential to reduce inputs and improve soil quality and crop yield, but there has been no research on the effect of AT on soil and wheat in a rice–wheat rotation system. In this study, field experiments were conducted to examine the effects of four tillage management methods (conventional tilling (CT) in each crop (RCT–WCT), no tilling (NT) in rice and conventional tilling in wheat (RNT–WCT, AT1), conventional tilling in rice and no tilling in wheat (RCT–WNT, AT2), and no tilling in each crop (RNT–WNT)) on the physical properties of soil, wheat growth, and yield. At the 0–5 cm soil layer, CT in the wheat season increased bulk density (BD) and decreased total properties, but it decreased BD at the 5–40 cm soil layer, and the effect of RCT–WCT was significantly greater than that of RNT–WCT. CT in the wheat season increased the root activity, root dry weight, net photosynthetic rate, leaf area index, antioxidant enzyme activities, and yield, and there was no significant effect between RCT–WCT and RNT–WCT. RNT-WCT has the potential to reduce inputs and maintain wheat yields

Theoretical and experimental study on a finite size vector sensor array with a cylindrically symmetric carrier

In this work, a finite size acoustic vector sensor (AVS) array is designed and its performance is theoretically and experimentally studied. The two-dimensional AVS array is comprised of five vector sensors and configured as a cross, and the array carrier is a cylindrically symmetric structure. Theoretical analysis and simulation indicate that the proposed method considering structure scattering can widen the working bandwidth. Furthermore, the utilization of vector sensor enables a significant white noise gain improvement at low frequencies, which makes the array more robust and easier to realize. Experiments have been done to study the array performance from several aspects including sensor noise characteristics, the beampattern, the direction-of-arrival (DOA) estimation ability and the array gain. From the change of the sensor directivity patterns or the amplitude distortion of the noise field, we can clearly observe the scattering field intensity. Moreover, it shows that the influence of the structure scattering on the tangential component of the vector field is symmetric, while that of the radial component is asymmetric. Experimental results also demonstrate that, with the proposed method, the 2nd and the 3rd order beamformers can be obtained which could be further used for the estimation of target DOA. In addition, an array gain of at least 6 dB is obtained capable of detection of weak signals. Our results indicate that the proposed array with a physical size less than one meter, although affected by nearby scatterers, can effectively break the Rayleigh limit and realize the remote detection in low-frequency regime