Bhattacharya Saikat, Reddy Divya, Reddy Raja, Sharda Asmita, Bose Kakoli, Gupta Sanjay
Epigenetics and Chromatin Biology Group, Gupta Lab, India.
Integrated Biophysics and Structural Biology Lab, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, MH, India.
Biotechnol Rep (Amst). 2016 Jul 18;11:62-69. doi: 10.1016/j.btre.2016.06.002. eCollection 2016 Sep.
Epigenetics have witnessed a renewed interest over the past decade and assays with recombinant histones has become an important tool for uncovering various aspects of histone biology. However, at times absence of recombinant histone accumulation in bacteria is encountered which is also commonly observed for many eukaryotic proteins in general. In this study, we have investigated the effect of multiple parameters on heterologous expression of proteins. We show that there is marked variability in the accumulation of H2A.2, H2B.1, H3.2 and H4 in the recombinant host, possibly owing to translational variability and degradation by the host proteases. We found that the variability could be overcome by incorporation of the commonly used purification tags, like GST or MBP, of appropriate size and position. Our results provide compelling evidence that transcript parameters like rare codon and GC content, mRNA secondary structure etc. together modulate translation kinetics and govern recombinant protein accumulation.
表观遗传学在过去十年中重新受到关注，使用重组组蛋白的检测方法已成为揭示组蛋白生物学各个方面的重要工具。然而，有时会遇到细菌中重组组蛋白积累缺失的情况，这在许多真核蛋白中也很常见。在本研究中，我们研究了多个参数对蛋白质异源表达的影响。我们发现，重组宿主中H2A.2、H2B.1、H3.2和H4的积累存在显著差异，这可能是由于翻译差异和宿主蛋白酶的降解所致。我们发现，通过引入常用的合适大小和位置的纯化标签，如GST或MBP，可以克服这种差异。我们的结果提供了令人信服的证据，即稀有密码子和GC含量、mRNA二级结构等转录本参数共同调节翻译动力学并控制重组蛋白的积累。
