Production of crystallizable fragments of membrane proteins.
Many membrane proteins feature autonomously folded extramembranous domains which, when isolated from the intact protein, perform biochemical functions relevant to biological activity. Whereas intact membrane proteins usually require detergent solubilization for purification, most extramembranous fragments are soluble in aqueous solution. If appropriately constructed, such fragments are often crystallizable and the resulting atomic structures can lead to important biological insight. In most instances, these fragments are produced in recombinant expression systems. To be crystallizable, molecular fragments should be uniform in composition and conformation and be available in abundance. Considerations for the production of crystallizable fragments of membrane proteins include the definition of fragment boundaries, the control of nonuniformities introduced by glycosylation of phosphorylation, and optimization of expression systems. These aspects are addressed here in general terms and in the case studies of applications to CD4, CD8, the insulin receptor kinase, and N-cadherin.
Hendrickson
W A
WA
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
D000818:Animals; D015704:CD4 Antigens / Q000302:isolation & purification; D016827:CD8 Antigens / Q000302:isolation & purification; D015820:Cadherins / Q000302:isolation & purification; D003460:Crystallization; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011972:Receptor, Insulin / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D015704:CD4 Antigens; D016827:CD8 Antigens; D015820:Cadherins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011972:Receptor, Insulin