Abstract
The mouse mammary tumor virus (MMTV) protease gene was cloned into pGEX-2T, an Escherichia coli expression vector containing the glutathione S-transferase coding region of Schistosoma japonicum. The chimeric protein was formed by fusion of the glutathione S-transferase with a hexapeptide which contains a thrombin cleavage site, followed by the MMTV protease. Affinity chromatography on a glutathione-Sepharose 4B column was used to isolate the chimeric protein. After thrombin cleavage, the glutathione S-transferase and the protease were separated by gel filtration chromatography on a Sephadex G-75 column. The overall yield of the protease purification procedure was about 1 mg of protease/liter of culture, and the specific activity was 380 pmol/min.micrograms of enzyme. Like other retroviral proteases, the MMTV enzyme was active as a dimer, showed maximum activity at pH between 4 and 6, and could be inhibited by pepstatin A and a phosphinic acid derivative HIV-1 protease inhibitor. Enzymatic characterization of this protease reveals its broad specificity, showing a clear preference for the oligopeptide substrate mimicking the cleavage site at the amino-terminal end of the capsid protein (kcat/Km = 9725.5 M-1.s-1). The chimeric protein was also an active dimer and showed a similar Km (17 microM) for such an oligopeptide, although its kcat was about 10 times smaller. Autocatalytic processing of the MMTV protease was observed after expression of clones containing the natural cleavage site, as it occurs at the amino-terminal end of the viral protease, instead of the thrombin-sensitive sequence.
Highlights
The mouse mammary tumor virus (MMTV)protease In themouse mammary tumor virus (MMTV),' the precurgene was cloned into pGEX-QT, an Escherichia coli sor polypeptides (Gag, Gag-Pro, and Gag-Pro-Pol) are assemexpression vector containing the glutathione S-trans- bled into immature cores (A particles) in the cytoplasm of ferase coding region of Schistosoma japonicum
Expression and Purification of the Fusion Protein andIsolation of MMTV Protease-The nucleotide sequence of the protease-encoding region of p202 was determined and found t o be identical to that observed in pPR207 and pPR14.6
In pPR207, the insert containing the protease gene was synthesized by polymerase chain reaction (PCR) reaction using the plasmid p202 as a template (Fig. 2), while the insert cloned in pPR14.6 was obtained after digestion of p202 with the restriction endonucleases DraI and BglII (Fig. L4).The identical sequences of p202, pPR207, and pPR14.6 showed 18 nucleotide changes when compared to that of the BR6 strain of MMTV, reported by Moore et al (1987), all but two of them were silent mutations
Summary
Vol 267, No., Issue of November, pp. 24134-24139,1992 Printed in U.S.A. Purification and Characterization tohfe Mouse Mammary Tumor Virus Protease Expressed iEnscherichia coZi*. After reveals its broad specificity, showing a clear prefer- thrombin cleavage of this chimeric protein, the MMTV proenceforthe oligopeptide substrate mimicking the tease was purified by gel filtration chromato~aphyand used cleavage site at the amino-terminal end of the capsid for determination of the kinetic parameters reported. The proteolytic activity recovered after different purification steps was measured by using as a substrate the synthetic dodecapeptide LTFTFPVVFMRR, which mimics the cleavage site found at the amino-terminal end of the capsid protein inMMTV. Kinetic parameters were obtained for a number of synthetic peptides mimicking the maturationsites inthe MMTV Gag polyprotein, as well as sites at theamino-terminal of the capsid protein in other retroviruses. The pBondapak C18 column (3.9 X 300 mm), and peaks were collected eluate from the affinity chromatography was concentrated as above and subjected to amino acid analysis for identification of the peptides
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
