Mozumder Sukanya, Mahesh Gopa, Srinivasan Krishnamoorthi, Sengupta Jayati, Mukherjee Sujoy
Structural Biology and Bioinformatics Division, CSIR Indian Institute of Chemical Biology, Kolkata, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India.
Bio Protoc. 2020 Aug 5;10(15):e3704. doi: 10.21769/BioProtoc.3704.
The serotonin 5-HT receptor (5-HTR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HTR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HTR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HTRs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.
血清素5 - HT受体（5 - HTR）是GPCR家族的成员，对各种神经功能很重要，其失调会导致许多心理健康障碍。对5 - HTR的结构研究需要产生从真核细胞培养物中表达的功能活性受体。在本方案中，我们描述了一种逐步方法，使用杆状病毒表达载体系统在源自我们对大鼠（匹配Uniprot ID P14842）和人类（匹配Uniprot ID P28223）5 - HTR研究的Sf9细胞培养物中表达和纯化血清素5 - HTR。该方法的一个独特之处是利用细胞培养添加剂以低感染复数感染细胞，从而与没有添加剂的先前方法相比，使用的病毒滴度数量减少了几倍。通过改变感染后收获时间，可以调整该方案以选择性地过度表达受体的糖基化或非糖基化形式。
