SEPARATION AND SEQUENCING OF FAMILIAR AND NOVEL MURINE PROTEINS USINGPREPARATIVE 2-DIMENSIONAL GEL-ELECTROPHORESIS

Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotide s for further molecular inquiry. In our laboratory, C3H10T1/2 murine f ibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformati on and carcinogenesis. Preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microsequencing. Isoelect ric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrot ransferred from 6 mm thick slab gels onto 0.22 mu polyvinylidene diflu oride membranes. Nearly 100 prominent blotted proteins were stained wi th Coomassie Brilliant Blue between pI 4.5-7.0 and 18-106 kDa and, of these, 27 prominent and well-resolved proteins were selected for seque ncing. Sequences of 14 to 24 amino acid residues in length were obtain ed from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functio ns while others had only recently been discovered, including a newly r eported Hsp 70 class member and a novel calcium-binding protein, retic ulocalbin. The new heat shock protein has a molecular mass of 75 kDa a nd has been designated as Grp75, PBP74, CSA or p66(mot-1) in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF-hand motif associated with high-affinity calcium-bin ding proteins. It may be involved in protein transport and luminal pro tein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteins. Thus, we have found that preparative 2-D PAGE serves as a powerful one-step purific ation method for protein isolation and characterization from an import ant in vitro murine model for the study of carcinogenesis.