Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes

Protein Eng. 2001 Jul;14(7):493-9. doi: 10.1093/protein/14.7.493.

Abstract

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cloning, Molecular
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / isolation & purification
  • Escherichia coli / genetics
  • Homeodomain Proteins / chemistry*
  • Homeodomain Proteins / isolation & purification
  • LIM Domain Proteins
  • Magnetic Resonance Spectroscopy
  • Metalloproteins / chemistry*
  • Metalloproteins / isolation & purification
  • Mice
  • Nuclear Proteins / chemistry
  • Protein Binding
  • Protein Engineering*
  • Protein Folding
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins
  • Transcription Factors / chemistry*
  • Transcription Factors / isolation & purification
  • Zinc / chemistry

Substances

  • Adaptor Proteins, Signal Transducing
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • LDB1 protein, human
  • LIM Domain Proteins
  • Ldb1 protein, mouse
  • Lmo2 protein, mouse
  • Lmo4 protein, mouse
  • Metalloproteins
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Zinc