Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli.

A J Zhang,G Bai,S Deans-Zirattu,M F Browner,E Y Lee

doi:10.1016/s0021-9258(18)45971-6

Abstract

The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.

Highlights

From the $Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami,Florida 33101 and the §Department of Biochemistryand Biophysics, University of California, San Francisco, California94143
The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography.Approximately20 mg ofpurified enzyme was reproducibly obtained from the cells derived from 2 liters of culture
Phosphorylase phosphatase, first discovered in 1943 (Cori and Green, 1943) has served as a paradigm for the study of the mammalian protein phosphatases.Its enzymology is complex and hasonly been unraveled during the passteveral years.Rabbit skeletal muscle and liver phosphorylase phosphatases were isolatedas proteins of about 35 kDa (Brandt et al, 1975a; Khandelwal et al, 1976; Gratecos et al, 1977); it was recognized at an early stage that these represented catalytic subunits associated with other regulatory subunits, based on the existence of larger complexes and theidentification of protein inhibitors of the enzyme (Brandt et al, 1974,1975b; Kat0 and Sato, 1974; Mellgren et al, 1979)

Summary

RESULTS

The resultantenzyme activity could be measured only in the Expression of Rabbit Muscle Phosphatase-1 as a n Insoluble Protein-Theinsertion of the codingsequencefor rabbit muscle ppase-1 into the PET vector led to expression of the protein as insoluble material in inclusion bodies which were clearly visible by light microscopy, similar to what has been observed for cathepsin D (Conner and Udey, 1990). 37-kDa polypeptide, could only be renatured to about 5% of reproducible in three preparations from cells isolated from 2 the expected specific activity (not shown). After heparin-Sepharose chromatography, second attempt a t expression the codingsequencewas in- the enzyme was almost pure as determined by SDS-PAGE; serted into the pTACTAC vector (see “Experimental Proce- several minor bands thawt ere present could be removed by a dures’’). Examination of the pTTZ-1 construct showed that second passagethrough heparin-Sepharose (notshown) withthis vector did allow the expression of ppase-1 in a soluble out a significantchangein specific activity. Thematerial form and that the expression of active enzyme was several obtained after a second heparin-Sepharose chromatography times higher when the cells were grown at 26-28 “C rather was found to be essentially homogeneous as shown, than at 37 “Cduring the induction phase (data not shown). This is identical with the behavior of authentic rabbit muscle ppase-1 (DeGuzman andLee, 1988; Brautigan et al, 1982) on SDS-PAGE it may be noted that the calculatedmolecular mass is 35 kDa (Bai et al, 1988)

Fraction Number

DISCUSSION

Expression of MPRhauobsscbpliethoPrhyolasspehatase