Temperature-sensitive production of rabbit muscle glycogen phosphorylase in Escherichia coli.
In order to understand how allosteric switches regulate both the catalytic activity and molecular interactions of glycogen phosphorylase, it is necessary to design and analyze variant proteins that test hypotheses about the structural details of the allosteric mechanism. Essential to such an investigation is the ability to obtain large amounts of variant proteins. We developed a system for obtaining milligram amounts (greater than 20 mg/l) of rabbit muscle phosphorylase from bacteria. Phosphorylase aggregates as inactive protein when a strong bacterial promoter is used under full inducing conditions and normal growth conditions. However, when the growth temperature of bacteria expressing phosphorylase is reduced to 22 degrees C we obtain active muscle phosphorylase. The degree to which the induced expression of phosphorylase protein is temperature sensitive depends on the strain of bacteria used. New assay and purification methods were developed to allow rapid purification of engineered phosphorylase proteins from bacterial cultures. The rabbit muscle phosphorylase obtained from the bacterial expression system is enzymatically identical to the enzyme purified from rabbit muscle. The expressed protein crystallizes in the same conditions used for growing crystals of protein from rabbit muscle and the crystal form is isomorphous. Rabbit muscle phosphorylase is one of the largest oligomeric mammalian enzymes successfully expressed in Escherichia coli. Our results indicate that optimization of a combination of growth and induction conditions will be important in the expression of other heterologous proteins in bacteria.
Browner
M F
MF
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0110.
Rasor
P
P
Tugendreich
S
S
Fletterick
R J
RJ
D000818:Animals; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors; D009132:Muscles / Q000201:enzymology*; D006005:Phosphorylases / Q000096:biosynthesis* / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013696:Temperature; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D006005:Phosphorylases