Falkenhagen Alexander, Asad Sabah, Read Stanley E, Joshi Sadhna
Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Canada.
Department of Pediatrics, The Hospital for Sick Children, Toronto, Canada.
BMC Biotechnol. 2016 Sep 2;16(1):66. doi: 10.1186/s12896-016-0288-3.
Recombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media.
Using a protein-based HIV entry inhibitor, soluble CD4 (sCD4), we demonstrated that 293 T cells transduced with a lentiviral vector mediated over 10-fold higher secretion of sCD4 in comparison to 293 T cells transfected with the corresponding plasmid. Secretion of sCD4 increased with the dose of the lentiviral vector up to a multiplicity of infection of 50. Exchanging the native signal peptide of sCD4 with the signal peptide of human alpha-1 antitrypsin increased expression by 50 %. There was no difference in expression from a monocistronic or bicistronic lentiviral vector. Reduction of the serum concentration in the culture media had no significant effect on the secretion of sCD4. Small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4. Purified sCD4 bound to recombinant HIV envelope glycoprotein 120 (Env gp120) and inhibited HIV entry at concentrations comparable to published results.
The procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. Furthermore, the method could be used to produce sCD4 fusion proteins or other His-tagged proteins.
用于治疗的重组蛋白理想情况下应在人细胞中生产，以确保适当的共翻译和翻译后修饰。然而，从培养基中纯化分泌蛋白受到转染细胞系低表达和血清蛋白存在的阻碍。在此，我们描述了一种基于慢病毒载体介导的基因递送和从人胚肾293T细胞中表达分泌型His标签蛋白，并直接从细胞培养基中进行亲和层析纯化的简单且经济高效的方法。
使用基于蛋白的HIV进入抑制剂可溶性CD4（sCD4），我们证明与用相应质粒转染的293T细胞相比，用慢病毒载体转导的293T细胞介导的sCD4分泌高出10倍以上。sCD4的分泌随着慢病毒载体剂量的增加而增加，直至感染复数达到50。将sCD4的天然信号肽与人α-1抗胰蛋白酶的信号肽交换可使表达增加50%。单顺反子或双顺反子慢病毒载体的表达没有差异。降低培养基中的血清浓度对sCD4的分泌没有显著影响。从50毫升血清含量降低的培养基中进行小规模纯化可产生高达1毫克的纯sCD4。纯化的sCD4与重组HIV包膜糖蛋白120（Env gp120）结合，并在与已发表结果相当的浓度下抑制HIV进入。
本研究中概述的程序无需特殊试剂或设备即可进行，任何实验室都可轻松采用。此外，该方法可用于生产sCD4融合蛋白或其他His标签蛋白。
