The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

家蚕，家蚕重组蛋白是生产重组蛋白的有前途的表达系统，但是由于存在大量宿主蛋白，纯化这些蛋白并不容易。为了研究纯化没有任何亲和标签的家蚕幼虫血淋巴中表达的重组蛋白的纯度，回收率和放大能力，我们使用红色荧光蛋白mCherry作为模型。热处理后，使用疏水色谱法（HIC），尺寸排阻色谱法（SEC）和肝素色谱法组成的三步色谱法可以大大减少宿主细胞的蛋白质含量。热处理对宿主细胞胞外蛋白的去除和纯度提高的影响最大。纯化后的样品中仍存在少量微量的宿主细胞蛋白，这表明从家蚕的血淋巴中纯化重组蛋白仍然具有挑战性。所提出的方案和亲和标签纯化分别使总蛋白质含量减少了99.84％和99.95％，而DNA的量分别减少了98.41％和99.53％。我们基于SDS-PAGE和毛细管电泳（CE）分析提出的方案的纯度分别为85.45％和43.60％，而Strep-tag亲和纯化的纯度分别为100％或63.69％。使用光密度测定法计算的总回收率为5.78％，高于使用Strep-tag亲和纯化法的4.09％。这项拟议的方案主要基于热处理，HIC，SEC和HiTrap肝素HP柱色谱法，