Apomyoglobin as a molecular recognition surface: expression, reconstitution and crystallization of recombinant porcine myoglobin in Escherichia coli.
Recombinant porcine myoglobin has been produced in Escherichia coli using the lambda cII fusion expression system of Nagai and Thøgersen [Nature, 309, 810-812 (1984)]. After processing and reconstitution with haem, the protein is gel-electrophoretically and spectrophotometrically indistinguishable from native pig myoglobin. Large crystals of both native and recombinant porcine myoglobin were grown from 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate. The crystals belong to space group C2 (a = 156.9 A, b = 42.0 A, c = 92.2 A, beta = 127.9 degrees) and diffract to a nominal 2.5 A resolution. We plan to explore apomyoglobin as a binding surface in studies combining site-directed mutagenesis and X-ray analysis. These experiments will be extended by studying the binding of haem analogues to the mutant apoproteins.
Dodson
G
G
Department of Chemistry, University of York, Heslington, UK.
Hubbard
R E
RE
Oldfield
T J
TJ
Smerdon
S J
SJ
Wilkinson
A J
AJ
D000818:Animals; D001059:Apoproteins / Q000378:metabolism*; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015394:Molecular Structure; D009211:Myoglobin / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013552:Swine
D001059:Apoproteins; D009211:Myoglobin; D011994:Recombinant Proteins; C010857:apomyoglobin