Cellular protein homogenates

The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10-15 per cent of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography. 

Topological proteomics means visualization of the abnormal cellular protein networks, cell by cell. Conventional proteomics is performed in three steps separation of complex protein mixtures, characterization of the separated proteins, and database searching to identify the composition of the complex. The employed techniques such as 2-D electrophoresis and mass spectrometry use tissue homogenates. Therefore, only quantitative changes of the most abundant proteins with particular biochemical properties can be detected. Any topological information, information on the cellular and sub-cellular distribution of proteins, that determines protein-interactions in networks, is lost. 

The p subunit was found in the pellet of the cell homogenate. After removal of cellular proteins by several washes with buffer solution the P subunit was solubilized in 4 M... 

We measured the cellular protein as an index of cell injury. After 3T3-L1 adipocytes were treated, the medium was aspirated and the cells were washed twice with ice-cold PBS and then scraped in 1 mL PBS. The cell suspension was homogenized using an ultrasonic homogenizer. The total protein in the supernatant was assayed using a Bio-Rad protein assay reagent (Bio-Rad Lab., CA, USA). 

Several researchers have tried to isolate cellular CBPs from the silkworm. In Nakajima s study (1963), the whole midgut mucosa was homogenized and the proteins separated with a gel-filtration chromatography column. Carotenoids were found in certain fractions containing proteins, suggesting the existence of CBPs in the midgut. Jouni and Wells purified a 35 kDa protein containing lutein... 

The cloned opiate receptors expressed in homogeneous cell lines associate with guanine nucleotide binding (G) proteins which couple the receptors to cellular effector systems. G proteins consist of hetero-trimeric complexes of a, fj and y subunits [66]. Multiple subtypes of each subunit have been identified and cloned. In particular, three subtypes of Gi have been cloned that are approximately 90% identical in amino acid sequence. Furthermore, two splice forms of G0 have been identified. 

An efficient cooling system minimizes protein denaturation (denaturation would otherwise occur due to the considerable amount of heat generated during the homogenization process). Ho-mogenizers capable of handling large quantities of cellular suspensions are now available, many of which can efficiently process several thousand litres per hour. 

Upon completion of the homogenization step, cellular debris and any remaining intact cells can be removed by centrifugation or by microfiltration. As mentioned previously, these techniques are also used to remove whole cells from the medium during the initial stages of extracellular protein purification. 

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