Characterization of recombinant and natural forms of the human LIM domain-containing protein FHL2.
FHL2 (Four and a Half LIM domain-containing protein 2) is a member of a small family of proteins with four LIM domains and an N-terminal half LIM domain. It is an intracellular protein thought to function as an adaptor in the formation of multi-protein complexes involved in signaling. To obtain human FHL2 in amounts allowing further characterization, we evaluated different expression systems and chose to express FHL2 with a His6 tag in insect cells using the baculovirus system. The recombinant protein was highly expressed and could be purified to >98% homogeneity as judged by SDS-PAGE analysis. Purified recombinant FHL2 was used to generate antibodies allowing detection and immunoprecipitation of FHL2 from human cells. Both recombinant and natural FHL2 were characterized by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular mass of the recombinant His6-tagged protein obtained by mass spectrometry was 36,995Da, in good agreement with the apparent mass of 36kDa in SDS-PAGE and slightly higher than the 35,981Da calculated from the sequence of the construct. The measured molecular mass of natural human FHL2 was 32,742Da and the calculated mass was 32,192Da. However, the apparent molecular mass in SDS-PAGE is 41kDa, indicating that the natural protein has an abnormal electrophoretic mobility. The results show that both the recombinant and the natural proteins are post-translationally modified and indicate that such modifications may lead to an abnormal electrophoretic behavior of natural human FHL2.
El Mourabit
Haquima
H
Center for Biochemistry, Faculty of Medicine, University of Cologne, Joseph-Stelzmann-Str. 52, Cologne 50931, Germany.
Müller
Stefan
S
Tunggal
Lucy
L
Paulsson
Mats
M
Aumailley
Monique
M
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D018398:Homeodomain Proteins; D006801:Humans; D060850:LIM-Homeodomain Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors
C105987:FHL2 protein, human; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors