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.

Eph/Ephrin memhrane proteins: A mammalian expression vector pTIg-BOS-Fc allowing rapid protein purification

There is an urgent need for high purity, single chain, fully functional Eph/ephrin membrane proteins. This report outlines the pTIg-BOS-Fc vector and purification approach resulting in rapid increased production of fully functional single chain extracellular proteins that were isolated with high purity and used in structure-function analysis and pre-clinical studies.

On-chip immunoprecipitation for protein purification

Immunoprecipitation (IP) is one of the most widely used and selective techniques for protein purification. Here, a miniaturised, polymer-supported immunoprecipitation (µIP) method for the on-chip purification of proteins from complex mixtures is described. A 4 µl PDMS column functionalised with covalently bound antibodies was created and all critical aspects of the µIP protocol (antibody immobilisation, blocking of potential non-specific adsorption sites, sample incubation and washing conditions) were assessed and optimised. The optimised µIP method was used to obtain purified fractions of affinity-tagged protein from a bacterial lysate.

Detergent-free membrane protein purification

Membrane proteins are localised within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilising or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilising protein structure is time consuming and expensive. Here we present a recently-developed method using a styrene maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification of a PHA-Like chitin-binding protein from Acacia farnesiana seeds: A time-dependent oligomerization protein

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI=4.0+/-0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al.

Purification, biochemical characterization and localization at single cell resolution of Matrix Gla Protein (MGP) and Bone Gla Protein (BGP) in the teleost fish Argyrosomus regius

Tese de dout. em Química, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2002

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Purification of the major group 1 allergen from bahia grass pollen, Pas n 1

Background Group 1 grass pollen allergens are glycoproteins of the β-expansin family. They are a predominant component of pollen and are potent allergens with a high frequency of serum IgE reactivity in grass pollen-allergic patients. Bahia grass is distinct from temperate grasses and has a prolonged pollination period and wide distribution in warmer climates. Here we describe the purification of the group 1 pollen allergen, Pas n 1, from Bahia grass (Paspalum notatum), an important subtropical aeroallergen source. Methods Pas n 1 was purified from an aqueous Bahia grass pollen extract by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography, and assessed by one- and two-dimensional gel electrophoresis, immunoblotting and ELISA. Results Pas n 1 was purified to a single 29-kDa protein band containing two dominant isoforms detected by an allergen-specific monoclonal antibody and serum IgE of a Bahia grass pollen-allergic donor. The frequency of serum IgE reactivity with purified Pas n 1 in 51 Bahia grass pollen-allergic patients was 90.6%. Serum IgE reactivity with purified Pas n 1 was highly correlated with serum IgE reactivity with Bahia grass pollen extract and recombinant Pas n 1 (r = 0.821 and 0.913, respectively). Conclusions Pas n 1 is a major allergen reactive at high frequency with serum IgE of Bahia grass pollen-allergic patients. Purified natural Pas n 1 has utility for improved specific diagnosis and immunotherapy for Bahia grass pollen allergy.

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Scale-up of fermentation and purification of recombinant allophycocyanin over-expressed in Escherichia coli

Phycobiliprotein is a photosynthetic antenna pigment found in cyanobacteria, rhodophytes, cryptophytes and certain dinoflagellates, which has been found to have anti-oxidative and anti-tumour activities. In this paper, a recombinant allophycocyanin (rAPC) had been expressed in Escherichia coli for anti-tumour effect. E. coli cells were cultured using glucose fed-batch method to achieve high cell densities. The biomass of rAPC was up to 3.52 g/L broth. The rAPC was purified from soluble E. coli cell lysate employing hydrophobic interaction chromatographic (HIC) method developed at the bench scale using 20 mL column. The process was performed at the pilot scale using 500 mL column for evaluation of scale-up. An amylose affinity column was used to improve the purity of final product in pilot scale purification. The purification process resulted in greater than 98% pure product and yielded up to 2.0 g/kg wet cells at the bench scale and 1.2 g/kg wet cells at the pilot scale. Peptide mapping was used to prove the identity of rAPC purified from bench scale and pilot scale process. Purified rAPC at the pilot scale was found to have remarkable inhibition on S-180 carcinoma in mice. (c) 2005 Elsevier Ltd. All rights reserved.

Pilot-scale fermentation and purification of the recombinant allophycocyanin over-expressed in Escherichia coli

A recombinant allophycocyanin (rAPC), used for treatment of tumors, has been expressed in E. coli which was grown in glucose fed-batch culture in a 30 l fermentor. Recombinant allophycocyanin was purified from soluble E. coli cell lysate using hydrophobic interaction chromatography followed by chromatography using amylose affinity column. The purity of product was greater than 98% and yielded an average of 5.5 g kg(-1) dry cells. Recombinant allophycocyanin significantly inhibited H-22 hepatoma (p (0.01) in mice with inhibition rates ranging from 36% to 62% with doses from 6.25 to 50 mg kg(-1) d(-1).