Expression, purification and mass spectrometric analysis of LIM mineralization protein-1 in human lung epithelial cells.
LIM mineralization protein-1 (LMP-1) is a novel osteoinductive protein that has been cloned and shown to induce bone formation both in vitro and in vivo. Detection and evaluation of the possible presence of carbohydrate structures in LMP-1 is an important regulatory consideration for the therapeutic use of recombinantly expressed protein. The sequence of LMP-1 contains a highly conserved N-terminal PDZ domain and three C-terminal LIM domains. The sequence analysis of LMP-1 predicts two potential N-glycosylation sites and several O-glycosylation sites. Here, we report the cloning and overexpression of LMP-1 in human lung carcinoma (A549) cells. Even though our group already reported the sequence of LMP-1 cDNA, we undertook this work to clarify whether or not the overexpressed protein undergoes any glycosylation in vivo. The expressed full-length recombinant protein was purified and subjected to chemical analysis and internal sequencing. The absence of any hexosamines (N-acetyl glucosamine or N-acetyl galactosamine) in chemical composition analysis of LMP-1 protein revealed that there is little or no post-translational glycosylation of the LMP-1 polypeptide in lung carcinoma cells (A549). We performed in-gel trypsin digestion on purified LMP-1, and the resulting peptide digests were analyzed further using matrix-assisted laser desorption and ionization mass spectrometry for peptide mass finger printing, which produced several exact matches with the corresponding LMP-1 peptides. Separation by high performance liquid chromatography and purification of the desired peptides followed by N-terminal sequencing resulted in many exact LMP-1 matches for several purified peptides, thus establishing the identity of the purified protein as LMP-1.
Sangadala
Sreedhara
S
Atlanta VA Medical Center and Department of Orthopaedics, Emory University School of Medicine, Atlanta, Georgia 30329, USA. ssangad@emory.edu
Titus
Louisa
L
Boden
Scott D
SD
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D060588:LIM Domain Proteins; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human; D011994:Recombinant Proteins