Culture supernatant proteins

The relatively small number of culture supernatant proteins (CSPs), compared with proteins in the cytoplasm, simplifies proteomic analysis. However, several aspects can make two-dimensional gel electrophoresis (2-DE) of CSPs challenging (6). These include (1) the presence of basic CSPs, which can become insoluble during isoelectric focusing, (2) the presence of hyaluronic acid, which can interfere with isoelectric focusing, (3) the presence of peptides in complex media, such as Todd-Hewitt broth, and (4) the abundance of specific exoproteins such as the cysteine protease SpeB, which can mask less abundant proteins (7). 

Fig. 1. Two-dimensional gel electrophoresis of culture supernatant proteins. Proteins were isolated from wild-type Streptococcus pyogenes strain NZ131 after 18 h of culture in 40 mL of chemically defined medium. Hundred micrograms of protein was separated with a 7-cm immobilized gradient strip (pH 4-7) (IPG GE Biosciences) and SDS-PAGE. Proteins were stained with SYPRO Ruby.

Lei, B., Mackie, S., Lukomski, S., and Musser, J. M. (2000) Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68, 6807-6818. 

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

Digestion of PGA by the PelL enzyme yielded a mixture of unsaturated ohgogalacturonides, giving evidence that PelL is an endo-deaving lyase (17). An exo-enz3mie, such as the EC 16 PelX, would generate a single product (15). The PelL protein differs from the major E. chrysanthemi pectate lyases in its ability to cleave both PGA and methylated pectin (17). The PelL activity has a basic optimum pH and an absolute requirement for Ca + ions. Analysis of culture supernatants demonstrated that PelL is an extracellular enzyme, such as the other secondary pectate lyases (17). 

A crude protein extract has been prepared by acetone precipitation on a three days old culture supernatant of the SCPP strain on Pg glc medium. In order to estimate the effect of glucose on PG activity, these protein extracts were deposited as dots on solid Pg glc medium. 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

The culture supernatant of K. marxianus was concentrated using PEG (20,000 Da) and the protein obtained was applied onto the column. Four peaks containing pectinolytic activities were resolved (Figure 1), and the molecular masses were calculated against markers as Mr of 47 kDa, 41 kDa, 35 kDa, and 33 kDa. 

VanAdrichem J.H.M., Bomson K.O., Conzelmann H., Gass M.A.S., Eppenberger H., Kresbach G.M., Ehrat M., and Leist C.H. (1998), Investigation of protein patterns in mammalian cells and culture supernatants by matrix assisted laser desorption/ionization mass spectrometry, Anal. Chem. 70, 923-930. 

Apply the hybridoma culture supernatant to the protein A column and monitor the eluent for protein at 280 nm. Wash the column with 30-40 mL of phosphate buffer, pH 8.0 (see Note 4). 

A series of supernatant transfer studies uncovered the fact that HC also produce NO, and in relatively large quantities (Curran et al., 1989). A supernatant generated from KC 8 hr after exposure to LPS and interferon-gamma (IFN-y) stimulated HC -NO biosynthesis and caused a simultaneous decrease in protein synthesis. As seen in the coculture model, a 6- to 8-hr delay in measurable NOj + N03 synthesis was seen in the HC exposed to conditioned KC supernatant. However, unlike KC or KC + HC where equimolar concentrations of citrulline were released into the culture supernatant, no increase in citrulline release paralleled the N02 + N03 synthesis by stimulated HC. It is most likely that the citrulline enters the HC urea cycle and is not secreted. 

Caprylic (octanoic) acid can be used to isolate mammalian IgG from serum, plasma, ascites fluid, and hybridoma culture supernatant by precipitation of non-IgG protein (3) (see Note 6). Other methods have been described in which caprylic acid has been used to precipitate immunoglobulin depending on the concentration used. The concentration of caprylic acid required to purify IgG varies according to species (see step 2 below). 

Screening. Primary screening is necessary to eliminate nonspecific hybridomas as soon as possible. Screening is also used to test the hybridoma culture supernatant for antibody reactivity and specificity. As an example, an Epstein-Barr virus associated protein is coated onto plastic ELISA plates. After incubation of hybridoma culture supernatant, secondary enzyme-labeled conjugate and chromogenic substrate, a colored product indicates a positive hybridoma. Alternatively, immunocytochemical screening can be used. It is preferable to test hybridomas when at least three-quarters of them are confluent. 

In an IPCR application developed by Furuya et al. [27], interleukin 18 (IL-18) was studied as an important protein in a number of immunological derangements. An indirect sandwich IPCR (Fig. 3D) was used for the detection of IL-18 in cell culture supernatants and serum samples. A quantitative study of low-level IL-18 was carried out beneath a serum concentration of typically 96 135 ng/L. In a systematic variation of assay conditions, the amount of biotinylated 227-bp DNA marker used in the Universal-IPCR protocol was identified as a source for nonspecific amplification. Following an optimization of DNA concentration and PCR cycle number, 2.5 pg/L IL-18 was detected. 

The A. saitoi carboxypeptidase cDNA was cloned downstream of a GDP promoter, and the resulting plasmid, pGCP13, was used to generate recombinant A. saitoi carboxypeptidase protein. No enzymatic activity was detected in the culture supernatant. We detected the A. saitoi carboxypeptidase activity of the extract obtained from yeast cells transfected with A. saitoi carboxypeptidase cDNA in forward orientation (pGCP13), although no activity was observed with the vector alone at pH 3.1. The recombinant A. saitoi carboxypeptidase activity... 

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