Document 0171
 DOCN  M9470171
 TI    Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse
       transcriptase gene to prevent dimerization of the expressed chimaeric
       protein: purification and characterization of a monomeric HIV-1 reverse
       transcriptase.
 DT    9409
 AU    Sharma SK; Basu A; Fan N; Evans DB; Upjohn Laboratories, Kalamazoo, MI
       49001.
 SO    Biotechnol Appl Biochem. 1994 Apr;19 ( Pt 2):155-67. Unique Identifier :
       AIDSLINE MED/94250350
 AB    We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant
       reverse transcriptase (RT) engineered to contain a 26-amino-acid linker
       insertion from the tether domain of feline leukaemia virus (FLV) RT. The
       chimaeric protein was expressed in Escherichia coli and migrated on
       SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT
       has been prepared by the coordinated applications of
       immobilized-metal-affinity chromatography and gel filtration on Superose
       12 columns. The monomeric nature of this chimaeric HIV-I RT was further
       characterized by cross-linking studies using disuccinimidyl suberate.
       The RNA-dependent DNA polymerase activity of the monomeric chimaeric
       HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These
       results support our recent studies on the monomeric polymerase domain
       (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal
       to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy,
       Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The
       inability of the monomeric chimaeric HIV-1 RT to display polymerase
       activity like that of the heterodimeric HIV-1 RT is attributed to a
       decrease in the processive rate of DNA synthesis (75%) and DNA binding
       (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase
       H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT.
       These results suggest that the linker insertion from FLV RT does not
       interfere with the RNAase H activity associated with the monomeric HIV-1
       RT.
 DE    Amino Acid Sequence  Chimeric Proteins/CHEMISTRY/GENETICS/ISOLATION &
       PURIF  Chromatography, Affinity  Chromatography, Gel  Cross-Linking
       Reagents  DNA Polymerases/METABOLISM  Electrophoresis, Polyacrylamide
       Gel  Escherichia coli/GENETICS  Human  HIV-1/GENETICS  Leukemia Virus,
       Feline/CHEMISTRY/ENZYMOLOGY  Molecular Sequence Data  Molecular Weight
       Polymers  Protein Engineering  Reverse
       Transcriptase/CHEMISTRY/*GENETICS/ISOLATION & PURIF  Ribonuclease H,
       Calf Thymus/METABOLISM  Succinimides/CHEMISTRY  Support, U.S. Gov't,
       P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).