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Illustration of the protein purification strategy based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease
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The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein...
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... Therefore, the isolation of proteins without affecting their shape, composition, and activity has continually been an issue in proteomics research [8]. With the improvement of generation, many new technologies have been implemented for protein separation, along with multi-column plate adapters (MCPA) [9], the cell-surface show primarily based on the SUMO-ulp1 system [10], aqueous 2-segment system (ATPS) [11], chromatography [11,12], and magnetic separation [7,13], etc. According to the residences of proteins, it's a growing fashion to split proteins to combine a couple of methods that can hold the hobby and shape of proteins higher and reap better resolution. ...
... Currently, this technique has been suggested as able to simplify protein purification. [10] reported the application of cell-surface display in protein purification founded on the cleavage of a SUMO-fused target protein by Ulp1 protease revealed on the surface of Escherichia coli cells as described above ( Figure 5). SUMO, a ubiquitin-like protein, has been used to improve target protein stability and solubility through N-terminal fusion [18,19]. ...
... The effectiveness of the SUMO-Ulp1 structure in protein purification can be archived using two different vectors: (i) the expression of SUMO-fused target protein on cell surfaces and (ii) the expression of Ulp1 protease on cell surfaces [10]. In this system, the Nterminal of a SUMO-fused target protein will be cleaved by the surface-displayed Ulp1 protease. ...
Protein purification is an ever-vital technique for academia and industry. This paper
mainly reviews and discusses one of the core components of proteomics—the latest advances in
separation technology for protein components—focusing on five different methods. The multicolumn plate adapter (MCPA) system is incredibly economical for protein treatment research to
purify samples. Because of the affinity of excess Ulp1 protease for SUMO fusion, excess protein
products may be obtained within half an hour using this method. Magnetic separation strategies can
offer a better protein purification process in the future because of a few advantages. The evaluation
established that the aqueous two-phase system (ATPS) technique is a cost-effective, time-saving
(30 min), and high-recuperation approach that can be scaled up for commercial purposes. Therefore,
the ATPS may be a viable single-step separation purification method, moving away from multi-step
purification such as the chromatography technique. Our review provides a technique capable of
efficient protein purification.
... According to preliminary results, the authors stated that a recovery rate of around 80% could be acquired [41]. Finally, amongst the latest research in the field, Zhou et al. [42] have recently documented the implementation of UF membranes (MWCO:30 kDa) to enhance the purity of the red fluorescent protein (mCherry). According to the authors' findings, most of the impurities were removed during UF, achieving a protein purity of >90%. ...
Pressure-driven membrane-based technologies, such as microfiltration (MF), ultrafiltration (UF), and nanofiltration (NF), have been successfully implemented in recovering different types of biomolecules and high-value-added compounds from various streams. Especially, UF membranes meet the requirements for separating specific bioproducts in downstream processes, e.g. monoclonal antibodies (mAbs), which are recognized as proteins produced mainly by plasma cells. According to the importance and functionality of the mAbs, their recovery is a current challenge with these bioseparations. Nevertheless, mAbs recovery using UF-assisted processes has been smartly performed over the last decade. To the best of our knowledge, there are no reviews of the reported developments using UF technology toward mAbs separation. Therefore, the goal of this paper is to collect and elucidate ongoing research studies implemented for the featured separation of mAbs and other biotechnological protein-type molecules (e.g. adenovirus serotype, extracellular vesicles, red fluorescent protein, cyanovirin-N, among others) via ultrafiltration-aided systems. The literature evidence (e.g. research papers, patents, etc.) has been analyzed and discussed according to the purpose of the study. Importantly, the relevant findings and novel approaches are discussed in detail. To finalize this document, the advantages, drawbacks, and guidelines in applying membrane-based techniques for such a recovery are presented.
... According to preliminary results, the authors stated that a recovery rate of around 80% could be acquired [41]. Finally, amongst the latest research in the field, Zhou et al. [42] have recently documented the implementation of UF membranes (MWCO:30 kDa) to enhance the purity of the red fluorescent protein (mCherry). According to the authors' findings, most of the impurities were removed during UF, achieving a protein purity of >90%. ...
Meat by‐products and co‐products, including poultry and fish wastes, represent a current challenge for the food processing industry due to their harmful impact on the environment. Membrane technologies, thanks to their intrinsic properties, represent an emerging tool for the valorization of these streams according to the principles of a sustainable circular economy. A review of significant applications of membrane‐based technologies, including pressure‐driven membrane processes and electro‐membrane processes, for the recovery of protein‐based compounds from meat by‐products is presented along with their trends and future potentials in the production of functional ingredients. Classic and recent development works are analyzed and critically discussed according to the relevant results in the field. Literature data clearly indicate that membrane technologies represent an efficient and environmentally friendly option for the separation, fractionation, and purification of bioactive compounds from different meat co‐products. The implementation of such processes as a way of recovering biomolecules also gives environmental benefits by reducing the organic load of both by‐ and co‐products. © 2021 Society of Chemical Industry (SCI).
Protein, being a fundamental biological macromolecule, assumes a critical function in various vital activities of cellular growth, heredity, reproduction, and other biological processes. Moreover, the utilization of recombinant proteins is extensive in the advancement of pharmaceutical materials, enzyme industrial implementation, and fundamental proteomics investigation. Effective purification methods are necessary for the production of recombinant proteins as well as natural proteins and peptides to ensure their efficient production. Various methods have been developed to improve protein purification, incorporating the recent advancements in protein separation technology. This review is focused on the sophisticated approach to the purification techniques of protein, peptide, and recombinant protein. The system known as the multi-column plate adapter (MCPA) proves to be highly cost-effective in the field of protein purification methods. The assessment has determined that the method of employing an aqueous two-phase system (ATPS) is an economically sound, and highly efficient approach in protein purification. Thus, the ATPS might represent a feasible approach to single-stage purification, deviating from the complex chromatography procedure. The chromatography strategies have been improved for the purification of peptides including batch liquid, continuous and semi-continuous chromatography. In the realm of recombinant protein purification, affinity purification presents several advantages compared to alternative methods. To produce, identify, and purify recombinant proteins from their host systems in large quantities with high efficiency, affinity strategies—particularly fusion strategies—have been developed. Hence, the purpose of this review is to implement the various purification technologies, their potential employments, fundamentals, advantages, and limitations.
Serratia marcescens nuclease (SM nuclease) can remove nucleic acid residues in recombinant protein drugs and reduce the viscosity of bacteria, exhibiting great significance in investigating these drugs. However, its underexpression in E. coli leads to less protein amount obtained by purification of inclusion body and low enzyme activity. In this study, the Small Ubiquitin-like Modifier (SUMO) tag was fused to the N-terminus of the SM nuclease and cloned into the pET28a vector. Subsequently, the expression and purification of inclusion body of the SUMO-fused SM nuclease were compared with those of SM nuclease without SUMO fusion. The results revealed that SUMO fusion elevated the expression of inclusion body of the SM nuclease, but exerted no effect on soluble expression of the protein. Meanwhile, SUMO fusion increased the solubility of inclusion body proteins and enhanced the removal of surface impurities during inclusion body washing. On the other hand, SUMO fusion promoted correct folding of the protein and improved the efficiency of refolding. The High-Performance Liquid Chromatography (HPLC) results indicated a protein concentration of 99% after two cycles of affinity chromatography for SUMO-fused SM nuclease. Additionally, the activity of the SUMO-fused protein (4×10 ⁶ U/mg) was 32 times higher than that of the unfused protein. SUMO fusion yielded approximately 10 mg of SM nuclease protein with a purity of 99% from 1 g of bacteria.
Protein fusion technology has had a major impact on the efficient production and purification of individual recombinant proteins. The use of genetically engineered affinity and solubility-enhancing polypeptide "tags" has a long history, and there is a considerable repertoire of these that can be used to address issues related to the expression, stability, solubility, folding, and purification of their fusion partner. In the case of large-scale proteomic studies, the development of purification procedures tailored to individual proteins is not practicable, and affinity tags have become indispensable tools for structural and functional proteomic initiatives that involve the expression of many proteins in parallel. In this chapter, the rationale and applications of a range of established and more recently developed solubility-enhancing and affinity tags is described.
Elevated expression of transforming growth factor β1 (TGF-β1) have been implicated in the pathogenesis of liver fibrosis, thus attenuating the excessive TGF-β1's activity by TGF-β1-binding peptide is an ideal strategy for the treatment of liver fibrosis. However, the application of small peptide as a pharmaceutical agent is obstacle due to difficult preparation and non-selectively deliver. The I-plus sequences of circumsporozoite protein (CSP-I) possesses high affinity for heparan sulfate proteoglycans, which are primarily located on liver tissues. TGF-β1-binding peptide P15 hold specific ability of binding to TGF-β1. In this study, we describe an approach to efficiently preparing liver-targeting peptide P15-CSP-I, which is conjugation of the sequences of P15 to the N-terminus of CSP-I, from the cleavage of biological macromolecule SUMO-tagged P15-CSP-I. In vitro and ex vivo binding assay showed that P15-CSP-I specifically targeted to the hepatocytes and liver tissues. Moreover, P15-CSP-I inhibited cell proliferation, migration and invasion, and decreased fibrosis-related proteins expression in TGF-β1-activated HSCs in vitro. Furthermore, P15-CSP-I ameliorated liver morphology and decreased the fibrosis responses in vivo. Taken together, P15-CSP-I may be a potential candidate for targeting therapy on liver fibrosis due to its high efficient preparation, specific liver-targeting potential and improved anti-liver fibrotic activity.
