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Membrane Protein Topology Determination by Proteolysis of Maltose Binding Protein Fusions by K.W. Miller; P.L. Konen; J. Olson; K.M. Ratanavanich is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Membrane Protein Topology Determination by Proteolysis of Maltose Binding Protein Fusions about?

A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest. Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain. A secreted MBP domain is attached to the protein if its \(\mathrm{N}\)-terminus resides in the periplasm. A cytosolic MBP domain (MBP \(\Delta 2-26\) ) is attached to the protein when its \(\mathrm{N}\)-terminus resides in the cytoplasm. The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first periplasmic loop. Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP. The mixture of digestion products is analyzed by Western immunoblotting using anti-MBP antiserum to detect cleavage fragments. Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain

Who reads Membrane Protein Topology Determination by Proteolysis of Maltose Binding Protein Fusions?

Author

K.W. Miller; P.L. Konen; J. Olson; K.M. Ratanavanich

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

1993

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)