High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata

Phycoerythrins have been widely used in food, cosmetics., immunodiagnostics and analytical reagents. An efficient one-step chromatography method for purification of R-phycoerythrins from Polysiphonia urceolata was described in this paper. Pure R-phycoerythrin was obtained with an absorbance ratio A(565)/A(280) of 5.6 and a high recovery yield of 67-33%, using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH, alternative to common gradient elution of ionic strength. The absorption spectrum of R-phycoerythrin was characterized with three absorbance maxima at 565, 539 and 498 mum, respectively and the fluorescence emission spectrum at room temperature was measured to be 580nm. The results of native-PAGE. and SDS-PAGE showed no contamination by other proteins in the phycoerythrin solution. which suggests an efficient method for the separation and purification of R-phycoerythrins from Polysiphonia urceolata. (C) 2004 Elsevier B.V. All rights reserved.

Genomic cloning, expression and recombinant protein purification of alpha and beta subunits of the allophycocyanin gene (apc) from the cyanobacterium Anacystis nidulans UTEX 625

A genomic fragment encoding alpha(APC) and beta(APC) (i.e., alpha and beta units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP ( maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. alpha(APC) and beta(APC) were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.

Purification and partial characterization of Mn superoxide dismutase from muscle tissue of the shrimp Macrobrachium nipponense

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

Isolation and purification of phycoerythrin from red alga Garcilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatogaphy

R-phycoerythrin was isolated and purified from Gracilaria verrucosa on an expanded-bed adsorption column combined with ion-exchange chromatography, which can effectively solve the problem of blockage of chromatographic columns due to polysaccharides during isolation and purification of phycobiliproteins. 0.1 M (NH4)(2)SO4 proved best to elute R-phycoerythrin from the expanded-bed column, and desalted 0.1 M (NH4)(2)SO4 eluate was used on an ion-exchange column to purify the R-phycoerythrin. Using this two-stage chromatography, the purity (OD565/OD280) of the R-phycoerythrin from G. verrucosa is increased to 4.4, and the yield of purified R-phycoerythrin can reach 0.141 mg . g(-1) of the frozen alga.

Large-scale isolation and purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed adsorption method

R-phycoerythrin, a light-harvesting protein in some marine algae, and can be widely used in medicine, was isolated and purified from a red alga, Palmaria palmata (Lannaeus) Kuntze, using the streamline column (expanded bed adsorption) combined with ion-exchange chromatography. Because the crude extract was applied to the column upwardly, the column would not be blocked by polysaccharides usually very abundant in the extract of marine alga, this kind of blockage could hardly lie overcome in ordinary chromatographic column. After applying the crude extract containing 0.5 mol/L (NH4)(2)SO4, (NH4)(2)SO4 solution of different concentrations (0.2 mol/L, 0.1 mol/L and 0.05 mol/L) was used to elute the column downwardly and the eluates were collected and desalted. The desalted eluates were then applied onto all ion-exchange chromatographic column loaded with Q-sepharose for further purification of the R-phycoerythrin. Through these two steps, the purity (OD565/OD280) of the R-phycoerythrin from P. palmata was up to 3.5, more than 3.2, the commonly accepted criterion for purity, and the yield of the purified R-phycoerythrin could reach 0.122 mg/g of frozen P. palmata, much higher than that of phycobiliproteins purified with the previous methods. The result indicated that the cost of R-phycoerythrin will drop down with the method reported in this article.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Purification of High-quality Plastid DNA from the Brown Alga Laminaria japonica Sporophytes

Extracting DNA from a variety of algae is rather difficult because of high levels of polysaccharides, tannins, and phenolics as these interfere with DNA isolation and downstream applications. High-quality plastid DNA (ptDNA) purification is particularly difficult because of its small proportion in total genomic DNA. This report describes an improved protocol for ptDNA purification that efficiently produces high-quality ptDNA from sporophytes of Laminaria japonica and several other algae. This improved protocol simplifies procedures for ptDNA purification and improves yield to 150-200 mu g of ptDNA per 100 g of frozen algal tissue. Polymerase chain reaction (PCR) amplification of conserved sequences has been used to verify purity of the ptDNA product.

Expression, purification, and characterization of recombinant Chinese shrimp crustin-like protein (CruFc) in Pichia pastoris

A crustin-like protein (CruFc) from Fenneropenaeus chinensis was expressed in Pichia pastoris and then purified to electrophoretic homogeneity on a Sephacryl S-100 column with a band corresponding to the expected one (13 kDa) shown by 15% SDS-PAGE. Western blot indicated that the rCruFc specifically reacted with polyclonal rabbit anti-Fenneropenaeus chinensis CruFc. Production in a 5 l bioreactor gave 237 mg rCruFc/l. Antimicrobial assay revealed that 4 mu M rCruFc inhibited growth of Staphylococcus aureus.

Heterologous expression and purification of recombinant allophycocyanin in marine Streptomyces sp isolate M097

Allophycocyanin is one of the most important marine active peptides. Previous studies suggested that recombinant allophycocyanin (rAPC) could remarkably inhibit the S-180 carcinoma in mice, indicating its potential pharmaceutical uses. Based on intergeneric conjugal transfer, heterologous expression of rAPC was first achieved in marine Streptomyces sp. isolate M097 through inserting the apc gene into the thiostrepton-induced vector pIJ8600. The transformation frequency for this system was approximately 10(-4) exconjugants/recipient. In the transformed Streptomyces sp. isolate M097, the yield of purified rAPC could amount to about 38 mg/l using a simple purification protocol, and HPLC analysis showed that the purity of the protein reached about 91.5%. In vitro activity tests also revealed that the purified rAPC had effective scavenging abilities on superoxide and hydroxyl radicals. This would widen the usefulness of the marine Streptomyces as a host to express the rAPC and to offer industrial strain for the production of rAPC.

Method for large-scale isolation and purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev

R-phycoerythrin was isolated and purified from a red alga, Polysiphonia urceolata Grev, using Streamline column combined with ion-exchange chromatography or hydroxyapatite chromatography. The purity of R-phycoerythrin isolated by Streamline column was up to 1.66 and the yield of R-phycoerythrin could be as high as 0.68 mg/g frozen P. urceolata. All the eluates from Streamline column were divided into two equivalent parts, respectively. One part was pumped into the ion-exchange column loaded with Q-Sepharose and the other was applied to the adsorption column loaded with hydroxyapatite. The purities of R-phycoerythrin purified using these two methods were both up to 3.26, more than 3.2 the commonly accepted criterion. The yield of purified R-phycoerythrin from the ion-exchange chromatography was 0.40 mg/g frozen P. urceolata and that from the hydroxyapatite chromatography could reach 0.34 mg/g frozen P. urceolata. The purified protein had three absorption peaks at 498, 535, and 565 nm and displayed a fluorescence maximum at 580 nm, which was consistent with the typical spectrum of R-phycoerythrin. The purified R-PE was also identified with electrophoresis. Only one single protein band appeared on native-PAGE with silver staining. SDS-PAGE demonstrated the presence of one 20 kDa major subunit, and one low intensity band corresponding to 33 kDa subunit. The results indicate that using the expanded bed adsorption combined with ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin can be purified from frozen P. urceolata on large scale. (c) 2006 Elsevier Inc. All rights reserved.

Purification and characterization of two types of chitosanase from a Microbacterium sp.

Two extracellular chitosanases (ChiX and ChiN) were extracted from Microbacterium sp. OU01 with Mr values of 81 kDa (ChiX) and 30 kDa (ChiN). ChiN was optimally active at pH 6.2 and 50 degrees C and ChiX at pH 6.6 and 60 degrees C (assayed over 15 min). Both the activities increased with the degree of deacetylation (DDA) of chitosan. ChiN hydrolyzed oligomers of glucosamine (GlcN) larger than chitopentaose, and chitosan with 62-100% DDA; but ChiX acted on chitosan and released GlcN. Hydrolysis of chitosan with 99% DDA by ChiN released chitobiose, chitotriose and chitotetraose as the major products.

Expression, purification of recombinant flounder MRF4 protein in Escherichia coli and analysis of its polyclonal antibodies

MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (130). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.

One step isolation and purification of liquiritigenin and isoliquiritigenin from Glycyrrhiza uralensis Risch using high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been successfully applied to the separation of bioactive flavonoid compounds, liquiritigenin and isoliquiritigenin in one step from the crude extract of Glycyrrhiza uralensis Risch. The HSCCC was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (2:2:1:0.6:2, v/v). Yields of liquiritigenin (98.9% purity) and isoliquiritigenin (98.3% purity) obtained were 0.52% and 0.32%. Chemical structures of the purified liquiritigenin and isoliquiritigenin were identified by electrospray ionization-MS (ESI-MS) and NMR analysis. (c) 2005 Published by Elsevier B.V.