Purification and biological characterization of bacterially expressed recombinant buffalo prolactin

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22?mg/L from 100?mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Purification of Monomeric Prolactin Charge Isoform from Buffalo Pituitaries

Abstract

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S‐200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti‐buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.     

Purification of monomeric prolactin charge isoform from buffalo pituitaries

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S-200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti-buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.     

Simultaneous isolation of prolactin and growth hormone from "discarded acid pellet" obtained from buffalo pituitaries

An acid pellet, obtained as a side fraction from a conventional gonadotropin purification pathway, has been found to contain the bulk of the pituitary lactogenic hormones (growth hormone or GH and prolactin or PRL). This discarded side fraction has been utilized to obtain buffalo lactogenic hormones (buGH and buPRL), simultaneously, and in bulk. The immunoreactivities of the purified semi-pure buffalo GH and PRL (APECS and APP-I, respectively) preparations were compared by direct binding ELISA with semi-pure standard buGH and PRL (ECS and EP-I, respectively) and were found to be as pure as standard semi-pure buGH and buPRL. When checked by direct binding ELISA using buGH and buPRL antisera, it was observed that APECS and APP-I were not cross-immunoreactive. SDS-PAGE and western blot analysis of APECS and APP-I showed major bands located at the same positions as in the case of standard semi-pure preparations (20 kDa for APECS and 23 kDa for APP-I). The semi-purified buGH and buPRL (APECS and APP-I) were converted to a highly purified preparation by chromatographing them via Sephacryl S-200 gel-filtration chromatography.     

Large-Scale Preparation of Recombinant Ovine Prolactin and Determination of Its in Vitro and in Vivo Activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb₂ cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

Expression, purification, and characterization of recombinant fibulin-5 in a prokaryote expression system

Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N²⁺-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.     

Cloning and Functional Expression of the Mucrosobin Protein, a β-Fibrinogenase of Trimeresurus mucrosquamatus (Taiwan Habu)

A venom-specific cDNA encoding for a thrombin-like enzyme designated as mucrosobin has been cloned and sequenced from the cDNA library of the venomous gland of Trimeresurus mucrosquamatus. The full-length cDNA of mucrosobin was assembled by oligonucleotide screening and 5′-rapid amplification of cDNA ends. The amino acid sequence deduced from the cDNA consists of 257 amino acid residues with a putative signal peptide of 24 residues. It is highly homologous to the other thrombin-like enzymes (batroxobin, mucofirase, and calobin), suggesting that it is a serine proteinase with a conserved catalytic triad of His⁴¹, Asp⁸⁴ and Ser¹⁷⁹ in the deduced form of mucrosobin protein. Northern blot analysis revealed that the mucrosobin gene encodes an mRNA of 1.5 kb and suggested a tissue-specific expression in the venomous gland. In an effort to study the biological property of mocrosobin, we have expressed the 28-kDa protein as inclusion bodies in Escherichia coli. For analyzing enzymatic activity, the inclusion bodies were solubilized and the recombinant protein was refolded with a two-step dialysis protocol. The refolded recombinant protein exhibited a specific β-fibrinogenolytic activity. This study offers a possibility of using genetic engineering to acquirie a functional snake venom protein with therapeutic potential.     

Expression, Purification, and Refolding of Active Recombinant Human E-selectin Lectin and EGF Domains in Escherichia coli

Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni²⁺ affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe^X (K _d = 321 nM, B_max = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.     

Expression and partial purification of human prolactin in Escherichia coli

1. Human prolactin has been expressed in Escherichia coli. A cDNA fragment coding for the signal sequence and the full length prolactin molecule was cloned into the expression vector pUR291 which directs the synthesis of a beta-galactosidase prolactin fusion protein when expressed in E. coli. 2. Cultures of E. coli harbouring the recombinant plasmid pJMBG62 produced a fusion protein of the appropriate molecular weight which was detected by Western blot analysis using a polyclonal antibody raised against pituitary-derived human prolactin. 3. The fusion protein was isolated from inclusion bodies in a partially pure form and it was used as immunogen to raise antibodies against human prolactin. 4. When this partially purified fusion protein was injected into rabbits it generated antisera with good prolactin titres in animals which were rested for one year following a disappointing primary immunization with purified human prolactin.     

Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli.

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 M) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both alpha-helical and beta-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization.     

Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed inEscherichia coli.The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256–257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)₆tag was expressed at high levels inE. colias inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni²⁺affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256–257 to remove 12 amino acids including the (His)₆tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein inE. coli.In addition, the refolded CMV PR could be crystallized for X-ray diffraction.     

Production of a polyclonal antibody against recombinant goldfish prolactin and demonstration of its usefulness in a non-competitive antigen-capture ELISA

The cDNA encoding the goldfish (Carassius auratus) prolactin was expressed in Escherichia coli using the pRSETA expression vector. The recombinant goldfish prolactin (gfPRL) produced was a fusion protein containing a hexahistidyl sequence, which facilitated its purification on a Ni²⁺ column. The fusion protein was overexpressed in the bacteria as inclusion bodies and was successfully purified under denaturing conditions by one-step affinity chromatography. Repeated immunization of rabbits against the purified recombinant gfPRL allowed the production of a high-titer polyclonal antiserum. The IgG fraction of the antiserum was isolated on an immobilized Protein A–agarose column. The antibody recognized recombinant gfPRL, but not recombinant goldfish growth hormone (gfGH) or goldfish somatolactin (gfSL) on Western analyses. The purified antibody was able to recognize gfPRL, but not gfGH or gfSL, in a non-competitive antigen-capture ELISA. The assay was applied in monitoring the purification of native PRL from goldfish pituitaries.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Inhibitory activity of the peptides derived from buffalo prolactin on angiogenesis

The peptide fragments obtained by cathepsin digestion of purified buffalo prolactin (buPRL) monomer have been characterized using SDS-PAGE and FPLC with regard to size and pI. Their antiangiogenic activity was tested in chick embryo chorioallantoic membrane (CAM) assay and the human endothelial cells wound healing assay. Antiangiogenic activity was observed in cathepsin-cleaved fragments from buPRL. Further, a peptide sequence 45A-46Q-47G-48K-49G-50F-51I-52T-53M-54A-55L-56N-57S-58C, which matched with human somatostatin (hSST), a known antiangiogenic factor, was located in the second loop between the first and second α-helices in the three-dimensional structure of buPRL, obtained by homology modelling. The synthetic peptide matching with SST sequence was found to exhibit antiangiogenic activity in both in vitro and ex vivo assays. It was also observed that all the peptides related to buPRL could antagonize the vascular endothelial growth factor (VEGF) and bradykinin (BK)-dependent production of endothelial nitric oxide (NO), which is a pre-requisite for endothelial tube formation. It is concluded therefore that an internal sequence in buPRL and peptide fragments derived from cathepsin-digested buPRL exhibit antiangiogenic activities.     

Overexpression, Purification, and Refolding of a Porphyromonas gingivalis Cysteine Protease from Escherichia coli

This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.     

Cloning, purification, and refolding of human paraoxonase-3 expressed in Escherichia coli and its characterization

Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and expressed in Escherichia coli. A majority of the expressed protein existed as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 and refolded in vitro. The refolded rhPON3 was purified by DEAE-Sepharose Fast Flow and its purity was up to 90%. The Km and Vmax values of refolded rhPON3, in respect to phenylacetate hydrolysis were 7.47 +/- 2.14 mM and 66 +/- 17 U/min/mg (n = 3). The Km and Vmax values of refolded rhPON3, in respect to dihydrocoumarin hydrolysis were 0.83 +/- 0.21 mM and 621 +/- 66 U/min/mg (n = 3). The refolded rhPON3 exhibited similar antioxidant activity to that of rhPON3 purified from the soluble fraction of cell lysate and could effectively protect LDL from Cu2+ induced oxidation.     

尖吻蝮蛇毒酸性磷脂酶A_2I的表达及其生化特征

将尖吻蝮蛇毒酸性磷脂酶 Ａ２ Ｉ（ Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ） 的基因克隆至表达载体ｐ Ｂ Ｌ Ｍ Ｖ Ｌ２ , 在大肠杆菌 Ｒ Ｒ１ 中成功表达。表达产物 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ约占细菌蛋白质总量的３０ ％ , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 Ｆ Ｐ Ｌ Ｃ Ｓｕｐｅｒｏｓｅ Ｔ Ｍ１２ 纯化, 产物经过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测只有单一条带。对表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 Ａ２（ Ａ Ｐ Ｌ Ａ２） 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 Ａ２（ Ｐ Ｌ Ａ２） 的结构与这些活性的关系进行了讨论     

尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A2 (A .aBPLA2 )基因克隆至温敏表达载体 pBLMVL2 ,在大肠杆菌RR1中成功诱导表达 .表达产物A .aBPLA2 约占细菌蛋白质总量的 2 0 % ,并以包涵体的形式存在 .纯化包涵体后 ,将产物变性、复性 ,然后用FPLCSuperoseTM12纯化 ,产物经过SDS 聚丙烯酰胺凝胶电泳检测只有单一条带 .对纯化后的表达A .aBPLA2 进行了酶活性、抑制血小板聚集活性和溶血活性的测定 .结果显示 ,表达A .aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2 酶活性相近 ,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2 的溶血活性 ,没有抑制血小板聚集活性 .最后对磷脂酶A2 的结构与这些活性的关系进行了讨论