Department of Physical and Analytical Chemistry, Jaén University, E.P.S. of Linares, Alfonso X El Sabio, No. 28, 23700 Linares, Jaén, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Mar 1;879(7-8):511-9. doi: 10.1016/j.jchromb.2011.01.013. Epub 2011 Jan 15.
In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90 mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90-93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.
本文开发了一种从蓝藻集胞藻中大规模纯化藻蓝蛋白（C-PC）的方法。通过渗透压休克从细胞中提取藻胆蛋白，并通过将离心后的细胞悬浮液通过膨胀床吸附色谱（EBAC）柱，使用 Streamline-DEAE 作为吸附剂进行分离。洗脱的富含 C-PC 的溶液最后通过使用 DEAE-纤维素的填充床色谱法进行纯化。使用磷酸盐 0.05 M 缓冲液 pH 7.0 进行两次优化提取。在 15mm 内径（ID）的小柱上对 EBAC 的操作进行了小规模优化。最佳条件为 4.9mg C-PC/mL 吸附剂的样品负荷、两倍于沉降床体积的膨胀床体积和 1.020mPa·s 的样品粘度。然后通过增加柱 ID（15、25、40、60 和 90mm）来放大 EBAC 工艺，并通过确定蛋白质突破容量和产物回收率来验证放大过程的成功。对于每个柱直径，EBAC 步骤的产率在 90-93%的范围内。为了获得纯 C-PC，利用传统的 DEAE-纤维素离子交换色谱法，产率为 74%。包括所有步骤的过程总产率为 69%。使用 SDS-PAGE 监测纯化步骤，并通过吸收和发射光谱法和反相高效液相色谱法（RP-HPLC）确认回收的 C-PC 的纯度。结果表明，EBAC 方法是一种可扩展的技术，允许在不损失产品的情况下获得大量的 C-PC，同时保持高蛋白质回收率，降低加工成本和时间。
