Purified proteins are important for determining structure. Today, the use of different protein expression systems has become the practice of many laboratories. The choice of expression system usually depends on the species, the degree of modification of the protein, and the yield of the protein of interest. The use of a signal sequence in an expression vector allows secretion of a relatively high-purity expression protein in a cell culture medium without the use of expensive cell disruption equipment, and can greatly shorten the time required to optimize the purification method [1]. . However, this method has a major drawback in that it takes a lot of time to clarify a large amount of cell culture fluid before purification. In addition, the centrifuge used has a limited throughput, which also increases the time required to clarify the medium as it must be run multiple times to meet our needs. The author also notes that when filtering insect cell culture media, the flow rate is still very slow even if the cells have been removed by centrifugation. This may be related to the presence of submicron particles, which remain suspended in the mammalian cell culture fluid after centrifugation, resulting in clogging of the filter membrane [2]. The Sartoclear Dynamics® range of filtration products was originally developed to greatly reduce the time required to filter mammalian cells from the culture. By adding diatomaceous earth to the culture solution, the porous filter cake can be molded, thereby preventing clogging of the filter, and rapidly obtaining the cell culture supernatant in the sample (Fig. 1). Since the centrifugation step is not required, problems due to centrifugal capacity and availability can be avoided. For the author's research, insect cells can express and secrete the target protein, and the culture supernatant must be purified. Therefore, the author tested the effect of Sartoclear Dynamics® on clarifying the culture supernatant of insect cells (containing the target protein) and compared the results with standard methods. Figure 1: Principle of clarifying cell culture using traditional methods and Sartoclear Dynamics® Lab bulk feed filtration Materials and Method An insect baculovirus expression system is used to express the target protein. Sf9 cells (Thermo Fisher Scientific, 11496015) were cultured in Sf-900TM II SFM (Thermo Fisher Scientific, 10902088) according to the manufacturer's instructions. A 1.5 L suspension culture solution was infected with a baculovirus harboring the gene encoding the target protein, and incubated at a temperature of 27 ° C for 24 hours with shaking at 240 rpm. The culture was harvested and divided into three 470 mL equal samples for clarification and protein purification. The first equivalent sample was clarified using standard methods. After the cells were centrifuged at 5,000 g for 15 minutes, the supernatant was passed through a 0.22 μm PES filter. The second and third samples were then clarified using the Sartoclear Dynamics® system. 5 or 10 g of diatomaceous earth was added, and the solution was mixed into a uniform suspension, and then the culture solution was filtered using a 0.22 μm PES filter. The protein of each clarified sample in the cell culture medium was purified using the Metal Complex Affinity Chromatography (IMAC) method. The medium was loaded in series onto a 5 x 1 mL HisTrap column (General Life Sciences, 17-3712-05) and pre-equilibrated using HTE buffer (50 mm HEPES, 500 mm NaCL, pH 7.4). The column was washed with 100 mL HTE buffer and 75 mL (containing 25 mm imidazole) in HTE buffer to remove impurities and the target protein was eluted with 25 mL (containing 500 mm imidazole) in HTE buffer. The time taken to clarify each cell culture fluid sample was recorded (Figure 2). Divide recording time into preparation time (ie measuring culture medium volume and balancing centrifuge tubes or adding diatomaceous earth for standard methods or Sartoclear Dynamics® methods), centrifugation time (standard method only) and filtration time (both methods) ). Figure 2: Comparison of clarification methods based on processing time. Sartoclear Dynamics® Lab greatly reduces the time required to clarify cell culture media. A clarified cell culture medium was prepared using standard centrifugation filtration methods, which took approximately 34 minutes. Although Sartoclear Dynamics®, optimized with 5 g of diatomaceous earth filtration, does not reduce filtration time relative to standard methods, omitting the centrifugation step means at least a 53% reduction in total preparation time. By increasing the amount of diatomaceous earth to 10g, the total preparation time can be reduced by 6 minutes, saving at least 82% of the time. In terms of throughput, the standard methods and filtration times using 5 g and 10 g diatomaceous earth filtration optimization methods are equivalent to flow rates of 2.35, 2.35 and 14.1 L/h, respectively. During the preparation of the sample using 5 g of diatomaceous earth, partial sedimentation of diatomaceous earth and cells was observed during the filtration. It is possible that early sedimentation of diatomaceous earth results in a certain reduction in flow rate. Obviously, most diatomaceous earth/cell mixtures can be resuspended by gently shaking the filter to bring the flow back to normal levels; however, when filtering higher cell density media, the specification of 10g diatomaceous earth should be more As appropriate. To test the effect of Sartoclear Dynamics® on protein yield, the protein in each sample should be purified using the IMAC method and the elution peak size compared (Figure 3). Preparing a sample with a Sartoclear Dynamics® system optimized for filtration with 10 g of diatomaceous earth produces an elution peak with higher absorbance, indicating the presence of more protein, compared to the elution peaks that occur with standard methods for preparing samples. In contrast, the standard method produces a wider elution peak (ie, a higher capacity), so using diatomaceous earth mixed samples does not substantially affect protein production. in conclusion Sartoclear Dynamics® Lab accelerates the clarification process in cell culture media. Using this filtration system eliminates the need for centrifugation, saves a lot of time, and significantly increases productivity and throughput. After mixing the samples with diatomaceous earth, there was no negative impact on protein yield. The system is available in a variety of models and can be used to clarify cell culture media in 15mL to 1L batches to meet a wide range of user needs. 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In this study, the author tested the Sartoclear Dynamics® Lab V500 as a new technology for rapid clarification of cell culture fluids prior to Sf9 cell expression recombinant protein purification. The protein expression vector carries a signal coding sequence to ensure that the expressed protein is secreted outside the cell. Therefore, the cells in the medium must be removed before the protein is purified, but it is time consuming to operate using conventional methods (centrifugation after filtration). In this article, the author demonstrates the powerful performance of the Sartoclear Dynamics® Lab filter by comparing it with traditional methods. Not only does it not affect protein production, it also saves a lot of time; it is the perfect choice for any lab that is working to increase the productivity and throughput of insect cell expression systems.
introduction
Understanding the three-dimensional structure of a protein is the key to understanding its function. For proteins involved in disease, three-dimensional structural information will help to design new, highly specific drugs for targeted targets.
Results and discussion
By harvesting the cell culture medium, it was found that the total cell density per ml was measured to be 1.5 × 10 6 . 
Figure 3: Samples were prepared using a standard centrifugal filtration method and a Sarcoclear Dynamics® Lab filtration system optimized with 10 g of diatomaceous earth to compare the protein elution peaks (★) between the two.
Improve the productivity of insect cell expression systems, it is the perfect choice
Summary