Document 0708
 DOCN  M9590708
 TI    Improving the Solubility Properties of HIV-1 Integrase
 DT    9509
 AU    Craigie R; Jenkins TM; Hickman AB; Ghirlando R; Laboratory of Molecular
       Biology, National Institute of Diabetes; and Digestive and Kidney
       Diseases, NIH, Bethesda, MD
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session III, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920011
 AB    Structural studies of retroviral integrase proteins have been obstructed
       by the poor solubility of these proteins. Our previous attempts to
       overcome this problem with HIV-1 integrase, including extensive trials
       of different solvent conditions and construction of deletion
       derivatives, have met with only limited success. The catalytic core
       domain was found to be relatively soluble under certain conditions in
       the presence of CHAPS detergent, but even under these conditions the
       multimeric state was heterogeneous. In addition, a carboxyl terminal
       fragment of integrase was relatively soluble. Attempts to crystallize
       these fragments were not successful. We next focussed our attention on
       the core domain, consisting of amino acids 50-212, because it is the
       minimal domain that is capable of polynucleotidyl transfer. We suspected
       that the insolubility of this domain may result from surface exposure of
       hydrophobic residues. In an attempt to overcome the problem, we
       constructed a set of mutant core domains in which one or more amino
       acids in every hydrophobic stretch was substituted with either lysine or
       alanine. In general, lysine was chosen for mutants containing a single
       amino acid substitution and the more conservative alanine mutation was
       used when more than one substitution was made. Each of the mutant
       proteins was expressed in E.coli. Lysates of induced cells were
       centrifuged and the solubility of the expressed protein was assayed by
       SDS-PAGE of the supernatants. One of the thirty mutant core proteins, a
       single amino acid substitution of lysine for phenylalanine at position
       185 (INF185K) was markedly more soluble than the wild type protein. This
       protein was purified and assayed for enzymatic activity and physical
       properties. IN F185K is at least as active as the wild type core in
       assays for disintegration activity. Gel filtration and equilibrium
       centrifugation analysis showed it to be a monodisperse dimer. The
       protein could be concentrated to greater than 25 mg/ml. It has been
       crystallized and the structure has been solved to 2.5A resolution (Dyda,
       F., Hickman, A.B., Jenkins, T.M., Engelman, A., Craigie, R., and Davies,
       D.R. (1994) Science 266, 1981-1986)
 DE    Alanine/GENETICS  Centrifugation  Chromatography, Gel  Cloning,
       Molecular  DNA Nucleotidyltransferases/*CHEMISTRY/GENETICS  Detergents
       Electrophoresis, Polyacrylamide Gel  Escherichia coli/GENETICS
       HIV-1/*ENZYMOLOGY  Lysine/GENETICS  Mutation  Solubility  MEETING
       ABSTRACT

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