Extended N-terminal sequencing of proteins of archaebacterial ribosomes blotted from two-dimensional gels onto glass fiber and poly(vinylidene difluoride) membrane

Previously uncharacterized proteins from intact ribosomes and ribosomal subunits of the extreme halophile Halobacterium marismortui (Haloarcula marismortui) were isolated and separated by high-resolution two-dimensional electrophoresis (2DE). N-Terminal amino acid sequences of 14 of these acidic large-subunit proteins were obtained by direct blotting of the separated proteins from two-dimensional electrophoresis gels to sequencer-stable supports followed by excision of the protein spots and sequencing. Furthermore, long internal sequences were obtained by in situ enzymatic cleavage of halobacterial proteins in gel pieces obtained from two-dimensional gels followed by electrophoretic separation of the fragments, blotting, and sequencing. Precautions are outlined for avoidance of N-terminal blockage of proteins, and the preparation and selection of suitable supports for obtaining extended N-terminal sequences are described. The results suggest that when prior fractionation is carried out to enrich for cell organelles, subcellular components of cells, or cell membranes, it is routinely possible to obtain numerous N-terminal sequences from one or a few 2DE gels of such fractions. Our results also indicate that, with appropriate precautions, proteins are routinely obtainable from 2DE gels in a form suitable for both N-terminal and internal sequence determination and show no detectable evidence for N-terminal blockage or destruction or modification of labile amino acid residues.     

Two-dimensional electrophoresis of proteins as a tool in the metabolic engineering of cell cycle regulation

Metabolic engineering of cell cycle regulation addresses important biotechnological problems about serum removal from animal cell culture systems. Chinese hamster ovary cells stimulated to grow by fetal calf serum, insulin, or basic fibroblast growth factor were studied by two-dimensional electrophoresis (2DE) and the resulting protein expression patterns were analyzed. Detailed 2DE protocols are provided and at least 24 gene products are identified which may play an important role in growth factor signaling. Moreover, a correlation between the expression of three proteins (cyclin D1, cyclin E, and E2F-1) and mitogenic strength was found. (c) 1996 John Wiley & Sons, Inc.