Madar David J, Patel Amar S, Lees Watson J
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.
J Biotechnol. 2009 Jul 15;142(3-4):214-9. doi: 10.1016/j.jbiotec.2009.05.003. Epub 2009 May 27.
The production of proteins using recombinant DNA technology often requires the use of in vitro protein folding. In order to facilitate in vitro protein folding, a redox buffer is added to the protein folding mixture. The redox buffer is composed of a small molecule disulfide and/or a small molecule thiol. Recently, redox buffers containing aromatic thiols have been shown to be an improvement over traditional redox buffers such as glutathione. For in vitro protein folding to be relevant to protein production on a larger scale, high protein concentrations are required to avoid large volumes of folding buffer. Therefore, we investigated the in vitro folding of lysozyme at 1 mg/mL instead of the traditional 0.1 mg/mL. Aromatic thiols and aromatic disulfides were compared directly with glutathione and glutathione disulfide, the most commonly used redox buffer. Folding experiments at pH 7 using aromatic thiols increased the yield by 20-40% and the folding rate constants by as much as 11 times relative to glutathione. At pH 8, improvements in yields of up to 25% and up to a 7-fold increase in folding rate constants were demonstrated. The effect of aromatic disulfide concentration was also investigated.
使用重组DNA技术生产蛋白质通常需要进行体外蛋白质折叠。为了促进体外蛋白质折叠，需向蛋白质折叠混合物中添加氧化还原缓冲液。该氧化还原缓冲液由小分子二硫键和/或小分子硫醇组成。最近，含有芳香族硫醇的氧化还原缓冲液已被证明比传统的氧化还原缓冲液（如谷胱甘肽）更具优势。为使体外蛋白质折叠与大规模蛋白质生产相关，需要高蛋白质浓度以避免大量的折叠缓冲液。因此，我们研究了溶菌酶在1mg/mL而非传统的0.1mg/mL浓度下的体外折叠情况。将芳香族硫醇和芳香族二硫键直接与最常用的氧化还原缓冲液谷胱甘肽和谷胱甘肽二硫化物进行了比较。在pH 7条件下使用芳香族硫醇进行的折叠实验，相对于谷胱甘肽，产率提高了20 - 40%，折叠速率常数提高了多达11倍。在pH 8时，产率提高了高达25%，折叠速率常数提高了高达7倍。还研究了芳香族二硫键浓度的影响。
