重组人心房利钠肽 (ANP) 的原核表达、纯化及鉴定

为提高重组人心房利钠肽(Atrial natriuretic peptide,ANP)的表达量,将3个ANP通过赖氨酸(Lysine,K)串联,并构建相对应的重组表达载体p ET28a(+)/ANP3。转染大肠杆菌进行诱导表达,目的蛋白约占菌体总蛋白的60%。经过包涵体变复性,赖氨酸酶(Lys-C)和羧肽酶(CPB)水解,以及一系列层析纯化,每升培养液可获得约16 mg的ANP蛋白。最终,纯化后的ANP经UPLC及Tricine SDS-PAGE鉴定,纯度大于90%,LC-MS鉴定显示其分子量为3 080 Da,且为二硫键正确形成的ANP单体,通过ELISA试剂盒检测,其具有和参比品一致活性。本研究为ANP的大规模制备打下了基础。同时,所采用的串联表达技术也为其他多肽类药物的重组表达提供了新的思路。     

Expression, purification and characterization of human urodilatin in E. coli

Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.     

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma: preferential increase of free ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP)

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma were analysed by gel permeation chromatography combined with a radioimmunoassay (RIA) for rat ANP (rANP). Gel permeation chromatography showed two immunoreactive peaks in rat plasma, one corresponding to alpha-rANP, rANP(99-126), and the other eluted at a high molecular weight, clearly different from gamma-rANP, rANP(1-126). The chromatographic profile of rat plasma after incubation with synthetic alpha-rANP demonstrated that the high molecular immunoreactivity had ANP-binding capacity. This bound form of ANP was almost totally excluded following extraction procedure, therefore, the immunoreactive ANP (ir-ANP) measured with the extraction assay was mainly free ANP. On the other hand, direct RIA may detect not only the free but also the bound form of ANP. Using both direct RIA and the extraction method, bound forms of plasma ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) were compared to normotensive Wistar Kyoto rats (WKY). Bound forms of plasma ANP in 20-week-old SHR and SHRSP were significantly higher than that in age-matched WKY. The ratio of free/bound form of plasma ANP in SHR and SHRSP also significantly increased compared to WKY, indicating a preferential increase in free ANP in the plasma of these hypertensive rats. These findings suggest that a bound form of ANP may be present in rat plasma and that it may play some pathophysiological role in the hypertension of SHR and SHRSP. Increased free ANP in plasma may indicate a compensatory increase in ANP release in these hypertensive rats.     

Characterization of specific binding of atrial natriuretic peptide (ANP) to rat PC12 phaeochromocytoma cells

Specific binding sites for atrial natriuretic peptide have been identified in membrane of the phaeochromocytoma cell line PC12. Scatchard analysis of binding studies revealed a Kd of 794 pM and a density (Bmax) of 254 fmol/mg protein. Hormones unrelated to ANP such as angiotensin II, bradykinin and arginine-8-vasopressin did not complete for the binding sites. Of the ANP-related peptides which competed for the binding sites, the following order of affinity was established; rANP (8-33) greater than rANP (28 amino acid) greater than rat atrial peptide fragment (13-28) greater than a-hANP (28 amino acid) greater than atrial peptide fragment (1-11) greater than atriopeptin I.     

Vascular receptor binding activities and cyclic GMP responses by synthetic human and rat atrial natriuretic peptides (ANP) and receptor down-regulation by ANP

Biological activities of a variety of synthetic human (h) and rat (r) atrial natriuretic peptide (ANP) and related peptides as assessed by receptor binding and cyclic GMP response, and regulation of vascular ANP receptors were studied in rat aortic vascular smooth muscle cells (VSMC) in culture. alpha-hANP1-28 and alpha-hANP7-28 equally inhibited the binding of 125I-labeled-alpha-hANP to its vascular receptors, whereas Met(O)12-alpha-hANP1-28 was less potent and reduced and carboxymethylated (RCM)-alpha-hANP1-28 was ineffective. rANP5-27 and rANP5-28 were equipotent in receptor binding, whereas rANP5-25 had somewhat less potent effect and rANP8-28 fragment was ineffective. alpha-hANP1-28, alpha-hANP7-28, rANP5-27 and rANP5-28 similarly stimulated intracellular cyclic GMP formation, whereas rANP5-25 showed less stimulatory effect, and RCM-alpha-hANP1-28, Met12-sulfoxide and rANP fragment were ineffective. Pretreatment with unlabeled alpha-hANP (3.2 X 10(-9) and 3.2 X 10(-8)M) for 24 hrs resulted in a substantial reduction (55 and 75%) of total receptor number without changing the affinity of ANP receptors. These results suggest that the common ring structure formed by the disulfide bond in the molecule is critical for receptor binding and subsequent biological actions, and that a hydrophobic amino acid located at the position of 12, and (24-26) residues at the C-terminal side, but not (1-6) at the N-terminal side, of the disulfide bridge may play a part in modulating receptor binding and/or biological functions. The present study also indicates "down-regulation" of vascular ANP receptors by homologous ligand.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Optimized procedures for producing biologically active chemokines

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6×His-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni–NTA–agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6×His-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni–NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.     

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.     

多串心钠素的纯化与活性测定

为获得纯化心钠素(ANP)单体,采用离子交换及疏水柱层析,纯化融合蛋白麦芽糖结合蛋白(MBP)-ANP和MBP-3ANP,用凝血因子Xa切割MBP-ANP后,经阳离子柱分离获得ANP单体.对ANP单体与BMP-3ANP进行生物学活性检测.1　材料与方法1.1　材料含心钠素多拷贝基因的重组表达质粒pMal-nANP...     

Affinity purification of recombinant interferon-alpha on a mimetic ligand adsorbent

A method for improved refolding and purification of recombinant human interferon-alpha (rh-IFN-alpha) from inclusion bodies is described. The optimal conditions of refolding were obtained by the addition of 0.5 M l-arginine to the refolding buffer. The rh-IFN-alpha was purified to near homogeneity utilizing a single-step chromatography on a mimetic dye-ligand matrix. Improved refolding, coupled to a single-column affinity purification strategy, resulted in a 10-fold increase in the yield of rh-IFN-alpha. This single-step purification protocol yielded approximately 50 mg of purified rh-IFN-alpha from 1 liter of shake flask culture. The rh-IFN-alpha prepared by this protocol was found to be essentially monomeric based on HPLC gel filtration and nonreducing SDS-PAGE. It had a specific activity of approximately 2.8 x 10(8) IU/mg, measured as inhibition of cytopathic effect of encephalomyocarditis virus on A549 human lung carcinoma cells.     

抗菌肽CP10A在大肠杆菌中的融合表达

CP10A是一种由抗菌肽Indolicine经过序列改造,且对多数革兰氏阳性病源细菌具有较强抗菌活性的多肽序列。本研究根据已报道的CP10A氨基酸序列,兼顾大肠杆菌密码子偏好性,设计CP10A的核苷酸序列,利用PCR技术合成相应的DNA序列,后克隆构建重组表达载体pET32a（+）-CP10A,转入大肠杆菌AD494菌株。经IPTG诱导表达和15% SDS-PAGE电泳检测后发现产物以包涵体形式存在,且融合表达量占总蛋白的50%。在变性条件下经Ni-NTA亲合柱层析及复性,最终获得了较高纯度的可溶性重组蛋白。本研究首次实现了CP10A抗菌肽在大肠杆菌中的融合表达,为进一步研究其生物学活性及应用奠定了一定的基础,同时也为研究抗菌肽表达提供了一种方法。     

Signal Peptide does not Inhibit Binding of Biotin to Streptavidin

Three recombinant polypeptides of streptavidin: the full-length streptavidin with a signal peptide (rsavS), full-length streptavidin (rsavF) and core streptavidin (rsavC), were expressed in E. coli strain BL21 (DE3) and purified by Ni-NTA chromatography. Although all three recombinant streptavidins had biotin-binding activity, the stability and solubility of rsavC tetraunits were much better than those of rsavS and rsavF, indicating that signal peptide and/or extra amino acid residues in rsavS and rsavF have negative effects on streptavidin. Meanwhile, the signal peptide and extra amino acid residues in rsavS and rsavF made it difficult for polypeptides to fold into functional proteins. After refolding of denaturing-purified proteins in vitro, both the specific activities and biotin binding sites of renatured streptavidins were 1.4-times as that of proteins obtained by native Ni-NTA purification. Because the denaturing-purified rsavC is easy of refolding into functional protein, the better strategy for production of active rsavC is to isolate the protein from IPTG-induced E. coli extracts by denaturing Ni-NTA affinity chromatography followed by refolding of purified polypeptide in vitro.     

Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor.

The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.     

An optimized system for expression and purification of secreted bacterial proteins

In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity chromatography, and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130 kDa yielded between 10 and 50 mg of fusion protein per liter of induced culture at greater than 90% purity, as judged by Coomassie-stained SDS-PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Hamaoka, B. Perman, A. Zemla, D.J. Leahy, J. Biol. Chem. 280 (2005) 17243-17250).     

Atrial Natriuretic Peptide Modulates Amiloride-Sensitive Na+ Transport Across the Blood-Brain Barrier

We obtained evidence that amiloride specifically potentiates 125I-labeled alpha-rat atrial natriuretic peptide (1-28) [atrial natriuretic peptide (ANP)-(99-126); rANP] binding to cerebral capillaries isolated from the rat cerebral cortex. The binding parameters, KD of 173 pM and Bmax of 159 fmol/mg of protein, became 33 pM and 88 fmol/mg of protein, respectively, when 10(-4) M amiloride was added to the incubation medium. When the effect of rANP was investigated on in vitro 22Na+ uptake into isolated cerebral capillaries, 10(-7) M rANP significantly inhibited the uptake in the presence of 1.0 mM ouabain, 1.0 mM furosemide, and 2.0 mM LiCl in the uptake buffer, a finding suggesting a specific inhibitory effect of rANP on amiloride-sensitive Na+ transport. Thus, the possibility that ANPs control amiloride-sensitive Na+ transport at the blood-brain barrier by interacting with specific receptors has to be considered.     

Effects of steroid and thyroid hormones on synthesis of atrial natriuretic peptide by cultured atrial myocytes of rat

The in vitro effects of various steroid and thyroid hormones on synthesis of rat atrial natriuretic peptide (rANP) were studied using new-born rat atrial myocytes in culture. Dexamethasone, testosterone and triiodothyronine markedly stimulated both synthesis and secretion of immunoreactive (IR)-rANP with the same peak after 4-day-culture. Dexamethasone and testosterone dose-dependently (10(-7)-10(-6) M) stimulated synthesis of IR-rANP and were the most potent among various steroids tested. Triiodothyronine (T3) also stimulated synthesis of IR-rANP in a dose-dependent manner (10(-8)-10(-7) M), of which effect was more potent than that of tetraiodothyronine, whereas reverse T3 was ineffective. The present study clearly shows that glucocorticoids, androgens and thyroid hormones directly stimulate synthesis of ANP by atrial myocytes and suggests that ANP may play a potential role in mediating and/or modulating the biological effects by these hormones in the cardiovascular system.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.