Robbins Puthenveetil1, Chul-Jin Lee1, Anirban Banerjee1. 1. Unit on Structural and Chemical Biology of Membrane Proteins, Neurosciences and Cellular and Structural Biology Division (NCSBD), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland.
Abstract
Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi- cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi- cells.
Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi- cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi- cells.
Authors: Samuel Wagner; Mirjam M Klepsch; Susan Schlegel; Ansgar Appel; Roger Draheim; Michael Tarry; Martin Högbom; Klaas J van Wijk; Dirk J Slotboom; Jan O Persson; Jan-Willem de Gier Journal: Proc Natl Acad Sci U S A Date: 2008-09-16 Impact factor: 11.205
Authors: Zachary E R Newby; Joseph D O'Connell; Franz Gruswitz; Franklin A Hays; William E C Harries; Ian M Harwood; Joseph D Ho; John K Lee; David F Savage; Larry J W Miercke; Robert M Stroud Journal: Nat Protoc Date: 2009 Impact factor: 13.491