Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.     

Cloning and recombinant expression of the La RNA-binding protein from Trypanosoma brucei

We report the isolation, cloning and recombinant expression of a Trypanosoma brucei homolog of the La RNA-binding protein. Based on peptide sequence information we have isolated a cDNA clone which encodes a protein of 335 amino acids with a predicted molecular weight of 37.7 kDa. The amino acid sequence fits the domain structure of known La proteins and contains a putative ATP-binding site located in the COOH-terminal domain. The cDNA was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and the recombinant protein displayed RNA-binding activity in an electrophoretic mobility shift assay.     

Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 M. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 & mgr;M. The E. coli expressed sNMT was homogenous and showed enzyme activity.     

Dictyostelium nucleomorphin is a member of the BRCT-domain family of cell cycle checkpoint proteins

A search of the Dictyostelium genome project database (http://dictybase.org/db/cgi-bin/blast.pl) with nucleomorphin, a protein that regulates the nuclear number, predicted it to be encoded by a larger gene containing a putative breast cancer carboxy-terminus domain (BRCT). Using RT-PCR, Northern and Western blotting we have identified a differentially expressed, 2318 bp cDNA encoding a protein isoform of Dictyostelium NumA with an apparent molecular weight of 70 kDa that we have called NumB. It contains a single amino-terminal BRCT-domain spanning residues 125-201. Starvation of shaking cultures reduces NumA expression by approximately 88+/-5.6%, whereas NumB expression increases approximately 35+/-3.5% from vegetative levels. NumC, a third isoform that is also expressed during development but not growth, remains to be characterized. These findings suggest NumB may be a member of the BRCT-domain containing cell cycle checkpoint proteins.     

Characterization of vitellin,egg-specific protein and 30 kDa protein from Bombyx eggs,and their fates during oogenesis and embryogenesis

Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

phyA基因新型表达质粒的构建及其在大肠杆菌中的高效表达

利用自行筛选、鉴定的黑曲霉F246,根据植酸酶基因(phyA)成熟肽编码序列设计引物,直接PCR扩增phyA,经酶切分析、DNA测序和氨基酸序列分析证实phyA基因克隆成功。从pMD18T-phyA克隆中获得phyA编码序列,将其与pET30a 质粒连接,构建pET30a -phyA重组质粒,并在大肠杆菌中获得了高效表达。重组质粒经IPTG诱导表达,SDS-PAGE特异区带分子量为50kDa,此重组蛋白占大肠杆菌可溶性蛋白的36.62％,酶活性较天然植酸酶高8倍以上。因此,该phyA基因具有正常的生物学功能,对其进行深入研究,为大量获得高活性植酸酶以及开发新型微生态制剂奠定了基础。     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

广西眼镜王蛇毒酸性磷脂酶A2-1在不同载体的表达

目的构建广西眼镜王蛇毒酸性磷脂酶A2-1(APLA2-1)在不同载体的重组表达质粒,在E.coli中表达APLA2-1并比较不同表达系统对APLA2-1的表达效果。方法将广西眼镜王蛇毒酸性磷脂酶A2-1(AP-LA2-1)基因克隆至表达载体pBLMVL2和pET28a( ),分别转化入大肠杆菌RR1和BL21,经过诱导表达,应用SDS-聚丙烯酰胺凝胶(SDS-PAGE)及Western blot观察重组蛋白表达情况。结果成功构建了重组质粒pBLMVL2-APLA2-1和pET28a-APLA2-1。pBLMVL2-APLA2-1在SDS-PAGE上没见明显表达带,在Western blot上可见一14 kD的表达带。pET28a-APLA2-1在SDS-PAGE上有一明显的18 kD表达条带,表达产物AP-LA2-1约占细菌总量30%,并以包涵体的形式存在。结论APLA2-1可在大肠杆菌中表达,pET28a( )对APLA2-1的表达效果优于pBLMVL2。     

Generate Western blot protein marker from a single construct

An expression construct, consisting of a tandem arrangement of nucleic acids coding for the constant fragments (Fc) receptor of protein G combined with nucleic acids for the Fc receptor of protein A, was constructed. When the construct was expressed in Escherichia coli, proteins of estimated molecular weights of 25, 30, 50, 58, 80, and 85 kDa were consistently obtained from this expression construct due to possible proteolytic degradation during the cultivation and purification steps. The purified proteins from this single expression construct were used as Western blot protein marker.     

A 35-kDa polypeptide of the crystalline lens in the common frog: its biochemical properties, tissue specificity and appearance in the developmental process

The vertebrate lens contains so-called taxon-specific water-soluble proteins. One of them is p-crystallin with a molecular weight of 35 kDa characteristic of Ranidae family. We have identified a polypeptide with a molecular weight of 35 kDa in the eye lens of Rana temporaria which: (1) can be extracted from the lens by aqueous salt solutions, (2) has a molecular mass of 36.1 +/- 0.4 kDa (by SDS-electrophoresis) and 37 kDa (by gel filtration), (3) is heterogeneous in terms of isoelectric point (pI 6.5-8.0), (4) binds to heparin-agarose, (5) denatures in response to freezing-thawing, lyophilization and in solutions with low ionic strength. Thus, major biochemical parameters of this polypeptide differ from that of amphibian alpha, beta- and gamma-crystallins. In addition to lens, 35 kDa polypeptide was detected by immunoelectroblotting in retina, testes, liver, kidney, spleen, stomach, intestine and lungs. Its level (as percentage of water-soluble protein) is 1.1 +/- 1.4% in the lens, 1.6 +/- 0.7% in retina. 0.05% in testes and liver and 0.01% or less in other organs. Thus, despite its wide tissue distribution, 53 kDa polypeptide is expressed predominantly in lens and retina. We studied the time-course of appearance and accumulation of this polypeptide in tissues where it is expressed at high or low levels. 35 kDa polypeptide was detected for the first time during larval development: (1) in the lens (some time after the mouth opening; stages 33-34 according to Dabagian and Sleptsova, 1975), (2) in the retina (by the time of anus opening; stages 36-37), (3) in the liver (at the stage of elongated hind limb bud; stages 40-41). Definitive expression level of this protein was achieved in the lens by the beginning of metamorphosis and in the retina and liver during first months of development. Hence, during the whole period of larval development 35 kDa polypeptide content of the lens exceeds that of retina or liver. A more substantial evidence is required to confirm the identity of studied polypeptide with rho-crystallin.     

家蚕抗菌肽CD高效表达及其抗BmNPV感染作用

通过大肠杆菌JM109诱导家蚕,提取其脂肪体总mRNA后,通过RT-PCR得到cDNA,根据GenBank上家蚕抗菌肽CecropinD的cDNA序列,设计并合成引物,然后PCR扩增得到CecropinD肽基因并克隆到pGEM-T载体中,经过EcoRΙ和XhoI酶切,连接并将CecropinD肽基因插入pET32a表达载体中。用重组质粒pET32a-ecropinD转化大肠杆菌BL21(DE3),在IPTG诱导下,融合蛋白Trx-CecropinD以可溶形式得到高效表达,经SDS-PAGE检测显示分子量为23kDa与预期大小相符,表达量约为总蛋白的30%。融合蛋白经Ni2 柱纯化后通过肠激酶切割后释放为Trx(18kDa)和CecropinD(5kDa),最后通过超滤管分离得到重组抗菌肽。通过抑菌实验测得重组CecropinD对于革兰氏阴性及阳性菌均有抑菌活性。并将重组CecropinD与家蚕病毒BmNPV作用混合4h后,一起投喂家蚕,发现病毒感染力有明显降低,说明其有抗病毒感染作用。     

Molecular response to TBT stress in marine sponge Suberites domuncula: proteolytical cleavage and phosphorylation of KRS_SD protein kinase

Marine sponges as sessile filter feeders are inevitably under a constant influence of changes in their environment. Mediation of extracellular signals and regulation of cellular response to environmental stress is a key function of cellular protein kinases. Expression, proteolytical cleavage and phosphorylation of stress-responsive KRS_SD protein kinase, in control and tributyl-tin (TBT) treated sponges were investigated. In control sponge, two KRS_SD proteins were expressed: KRS_SD1 (54 kDa) corresponding to KRS_SD calculated molecular weight, and KRS_SD2 (50 kDa). Exposure of sponges to TBT resulted in alteration of KRS_SD1 and KRS_SD2 expression levels and their phosphorylation state. KRS_SD1 band disappearance after 4 h coincides with the appearance of additional 35 kDa immunoreactive protein band (p35) and decrease in number of viable cells. Incubation of recombinant KRS_SD with protein extracts from TBT stressed sponges resulted in cleavage of recombinant KRS_SD protein. The size of the resulting protein fragment suggests caspase-3 recognition site, present in both recombinant KRS_SD and KRS_SD, as a possible site of cleavage. Phosphorylated KRS_SD bands appear at the same time as p35 and remain after p35 band intensity reached control level. KRS_SD induction, its phosphorylation and proteolytical cleavage during TBT stress suggest that in sponge cells exist mechanism similar to one present in human cells where KRS/MST protein kinase is involved in promotion of apoptosis following oxidative stress.     

Chrysoptin is a potent glycoprotein IIb/IIIa fibrinogen receptor antagonist present in salivary gland extracts of the deerfly

Salivary gland lysates of the deerfly (genus Chrysops) contain chrysoptin, an inhibitor of ADP-induced platelet aggregation, which presumably assists the fly in obtaining a blood meal. Chrysoptin has now been isolated, and its cDNA has been cloned and expressed. Chrysoptin was purified to homogeneity using anion exchange and hydrophobic interaction chromatography and found to be a protein with a molecular mass of 65 kDa as determined by gel electrophoresis. N-terminal amino acid sequencing allowed for the synthesis of degenerate oligonucleotides that led to cloning, from salivary gland specific mRNA, of the cDNA encoding this platelet inhibitor. No RGD sites are present in the predicted sequence. A search of GenBank(TM) did not reveal significant sequence homology between chrysoptin and other proteins. The molecular mass predicted from the cDNA was 59 kDa. Predicted glycosylation and phosphorylation sites may account for this difference in molecular mass, as recombinant chrysoptin expressed in Sf21 cells had a molecular mass of 65 kDa, matching that of the natural protein. Chrysoptin functions by inhibiting the binding of fibrinogen to the fibrinogen/glycoprotein IIb/IIIa receptor on platelets with an IC(50) of 95 pmol. These results reveal that insect salivary glands are a source of fibrinogen receptor antagonists.     

Isolation of the epithiospecifier protein from oil-rape (Brassica napus ssp. oleifera) seed and its characterization

The epithiospecifier protein (ESP) is a myrosinase (MYR) cofactor, which is necessary to drive the MYR-catalyzed hydrolysis of some specific glucosinolates towards the production of cyanoepithioalkanes instead of isothiocyanates and nitriles. ESP was isolated from Brassica napus seeds by anionic exchange and gel filtration chromatography. ESP showed a molecular weight of about 39 kDa and pI 5.3. The amino acid sequence of several tryptic peptides of ESP (accounting for about 50% of the total sequence) made it possible to establish the high similarity (81% identity) with a hypothetical 37 kDa protein (TrEMBL data base accession number Q39104) and several jasmonate-inducible proteins from Arabidopsis thaliana. This observation suggests that ESP is likely to be involved in jasmonate-mediated defence and disease resistance mechanisms.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Water relations and osmotic adjustment in Apium graveolens during long‐term NaCl stress and subsequent relief

Pea plants ( Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.     

Molecular cloning of a full-length cDNA for human alpha-N-acetylgalactosaminidase (alpha-galactosidase B)

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.