Culture filtrate proteins

Roberts, A.D., Sonnenberg, M.G., Ordway, D.J., Fumey, S.K., Brennan, PJ, Belisle, J.T., and Orme, I.M. 1995, Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis. 

Sonnenberg, M.G. and Belisle, J.T. 1997, Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infection Immunity 65 4515-4524. 

Wedlock, D.N. Keen, D.L. McCarthy, A.R. Andersen, P. Buddie, B.M. (2002). Effect of different adjuvants on the immune responses of Mycobacterium tuberculosis culture filtrate proteins, Vet Immunol Immunopathol, 86, 79-88. 

Hubbard RD, Floiy CM, Collins FM. Immunization of mice with mycobacterial culture filtrate proteins. Clin Exp Immunol 1992 87(l) 94-98. 

Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures. 

SDS gel electrophoresis separation in total denaturing conditions was carried out on the protein of culture filtrates and proteins of known molecular mass. The four dark bands (Figure 2) which appear in the gel between 45 and 36 kDa of the standards were assumed to be PG based on the gel filtration results for PG activity and total protein. The relative molecular mass of the four protein bands were estimated as 45 kDa, 42 kDa, 39 kDa and 36 kDa. It was calculated that about 85% of total protein secreted into the culture medium by K. marxianus consisted of PG. 

Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 fim nitrocellulose membranes. After transfer of proteins, membranes were... 

The policlonal antibodies raised against the main band of PG also reacted with the 68 kDa band mentioned above. Total proteins from culture filtrates were obtained from both conditions and analyzed by SDS-PAGE. The gel was subsequently blotted and the filter hibridized with the PG antibodies (Fig-3). The results confirmed the presence of a extracellular PG with similar migration in inducing and non-inducing conditions. 

The strains were cultured on Mandels medium + 1% citrus pectin for 5 days and the enzymatic activities of culture filtrates were determined on three substrates citrus pectin, polygalacturonic acid and filter paper, (a) extracellular proteins are in p.g/ml. (b) p>ectinolytic activities on pectin (PC) and on polygalacturonic acid (TO) and Pectin esterase (PE) are in units/ml. (c) total cellulolytic activity (filter paper, fp) are in mg of liberated reducing sugars/ml. 

Feruloyl esterase activity was first detected in culture filtrates of Strepto-myces olivochromogenes (49), and has thereafter also been reported for some hemicellulolytic fungi (Table III). A partially purified feruloyl esterase from S. commune liberated hardly any ferulic acid without the presence of xylanase (65). Very recently a feruloyl esterase was purified from Aspergillus oryzae (Tenkanen, M. Schuseil, J. Puls, J. Poutanen, K., /. Biotechnol, in press). The enzyme is an acidic monomeric protein having an isoelectric point of 3.6 and a molecular weight of 30 kDa. It has wide substrate specificity, liberating ferulic, p-coumaric, and acetic acids from steam-extracted wheat straw arabinoxylan. 

Figure 1. Fractionation of proteins in the culture filtrate of Trichoderma reesei according to their pi values Xyl, xylanase Ara, arabinosidase AE, acetyl esterase / X, /3-xylosidase aG, a-glucuronidase / G, / -glucosidase CBH, cellobiohydrolase EG, endoglucanase. Chromatofocusing was performed in a PBE-94 anion exchange resin (Pharmacia) with a pH-gradient created by ampholyte buffers (Pharmacia). Solid line, A dotted line, pH. (Reproduced with permission from ref. 24. Copyright 1988.)...

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity."0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

From a culture filtrate of B. subtilis strain F-ll, Kaji and Saheki4 purified endo-L-arabinanase to a homogeneous protein by hydroxylapatite... 

Unclassified strains gifts from Dr. J. H. Walsh, Department of Biochemistry, University of Manchester, Institute of Technology. Cellulase assays (21) were carried out for 7 days at 37°C with 1 mL of cell-free culture filtrate. Ci added amounted to 180-200 fig protein. 

In previous work, we obtained several cellulase components from culture filtrates of Irpex lacteus (Polyporus tulipiferae) or from Driselase, a commercial enzyme preparation of this fungus they behaved practically as a single protein (1,2,3). They were different in randomness of the hydrolysis of carboxymethyl cellulose (CMC), expressed as the ratio... 

Extracellular Cellulolytic Activities. The appearance of the CM-cellulase activity in a culture of Thermoactinomyces grown on 1% microcrystalline cellulose is shown in Figure 2. The extracellular CM-cellulase activity approached a maximum of 14-16 mg reducing sugar (RS) mL-1 min"1 within 18-24 hr. The Avicelase activity of the culture filtrate developed simultaneously with the CM-cellulase activity and amounted to 3 mg RS mL"1 hr"1. The extracellular protein concentration reached 1.7 mg/mL in the stationary phase (6). 

Figure 4. Preparative isoelectric focusing in a granulated gel of a desalted lyophilized culture filtrate (50 mg protein) from the late exponential growth phase of Thermoactinomyces sp. Separation carried out for 48 hr at a constant power of 8 W (29). (C>) CM-cellulase activity (A) Avicelase activity (6).

Figure 5 also shows two 10-hr samples, 10a and 10b. Sample 10a was stored in solution at 4°C for one week, while sample 10b was stored frozen and then thawed immediately before application to the polyacrylamide gel. Both samples show the same protein band pattern. If proteolytic enzymes in the culture filtrate had acted on and partially degraded the extracellular proteins, a different band pattern would have been expected. Thus no product-precursor relationship appeared to exist between the various extracellular proteins in a culture filtrate of Thermoactinomyces. Moreover, it seems as if this organism produces at least three different extracellular cellulolytic enzymes simultaneously. 

The two genes encoding ECB deacylase subunits from A. utahensis were cloned and overexpressed by S. lividans as soluble extracellular proteins [25], The recombinant S. lividans was grown in a 10-liter fermenter under conditions described previously [30], After 2 days of growth, the culture was filtered through 1 Whatman filter paper. The recombinant ECB deacylase from the culture filtrate... 

Mercille S, Johnson M, Lemieux R, Massie B (1994), Filtration-based perfusion of hybridoma cultures in protein-free medium - reduction of membrane fouling by medium supplementation with DNAse-I, Biotechnol. Bioeng. 43 833-846. 

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