Complexes of telomeric oligonucleotides with the PGEk protein vector: Internalization by target cells and antiproliferative activity

Recombinant protein PGEk, consisting of the human epidermal growth factor (hEGF) and a DNA-binding domain, proved to be capable of interacting with the hEGF receptor and inducing cell proliferation, as characteristic of hEGF. PGEk complexes with the telomere-mimic oligonucleotide d(TTAGGG)₄ (TMO) and its thio analog (TMS) were efficiently and selectively internalized by cells with a high-level expression of the hEGF receptors. The extent of internalization was studied as a function of the PGEk: oligonucleotide ratio in the complex. The intracellular location of the oligonucleotides was determined. PGEk was found to ensure a more efficient delivery of the oligonucleotides and to protect them from nuclease degradation. The oligonucleotides contained in the complexes exerted a far greater cytotoxic effect as compared with the free oligonucleotides.     

Complexes of telomeric oligonucleotide d(TTAGGG)4 with the new recombinant protein vector PGEk carrying nucleic acids into proliferating cells

A study was made of the complexation of the protein vector PGEk, which transfers nucleic acids into the nuclei of cancer cells, with phosphodiester d(TTAGGG)₄ (TMO) and phosphorothioate Sd(TTAGGG)₄ (TMS) oligonucleotides, which inhibit telomerase. PGEk (64 amino-acid residues) contains a hydrophobic domain that originates from the human epidermal growth factor (hEGF) and is responsible for the receptor-mediated transfer of PGEk across the cell membrane, and the hydrophilic domain, which is a nuclear localization signal (NLS) and serves to bind DNA and deliver it to the cell nucleus. Experiments were performed in 0.01-M Na-phosphate and 0.1-M NaCl at 37°C. An analysis of the circular dichroism (CD) spectra showed that TMO forms an antiparallel G-quadruplex, while TMS occurs in the form of unfolded strands. The number of PGEk molecules adsorbed on oligonucleotides was estimated from the quenching of PGEk fluorescence and the increase in its polarization upon titration with oligonucleotides. Adsorption isotherms were plotted in Scatchard coordinates. Adsorption of the first two PGEk molecules on TMO and TMS followed a noncooperative mechanism and was characterized by high association constants: K _1(TMO) = (7 ± 1) · 10⁷ M^?1 and K _1(TMS) = (3 (± 0.5) · 10⁷ M^?1. Further adsorption, up to five or six PGEk molecules per TMO molecule, showed high cooperation and K _2(TMO) = (4.0 ± 1.5) · 10⁶ M^?1. Unlike TMO, TMS only weakly bound the third PGEk molecule: K _2(TMS) = (8 ± 2) · 10⁵ M^?1. An analysis of the CD spectra showed that PGEk partly unfolded the G-quadruplex formed by TMO and did not have an effect on the single-stranded structure of TMS. The secondary structure of DNA and the number of protein subunits were established for the biologically active complexes PGEk-TMO and PGEk-TMS, which efficiently pass across the membrane of cancer cells and inhibit their proliferation.     

Complexes of telomeric oligonucleotide d(TTAGGG)4 with the new recombinant protein PGEk--nucleic acid carrier into proliferating cells

The complexation of the new protein vector PGEk--a carrier of nucleic acids into proliferating cells with phosphodiester d(TTAGGG)4 (TMO) and phosphorothioate Sd(TTAGGG)4 (TMS) telomerase inhibitor oligonucleotides was studied. PGEk molecule, consisting of 64 amino acids, is comprising the sequence of the human epidermal growth factor EGFh which is hydrophobic cell targeting moiety serving for receptor-mediated endocytosis and an NLS (nuclear localization signal) which is hydrophilic serving as a DNA-binding and selective nuclear import moiety. Experiments were carried out in 0.01 M Na-phosphate buffer contained 0.1 M NaCl, pH 7.8 at 37 degrees C. CD spectral analysis revealed that TMO molecules folded back into intramolecular antiparallel G-quadruplex while TMS molecules were represented as unstructured thread. The number of adsorbed PGEk molecules were estimated using PGEk intrinsic fluorescence decrease and fluorescence polarization increase of PGEk under oligonucleotide titration. Adsorption isotherms were plotted in Scatchard coordinates. We have shown that adsorption of the first two PGEk molecules on TMO and TMS occurs noncooperatively with the high association constants K1(TMO) = (7 +/- 1) x 10(7) M(-1) and K1(TMS) = (3 +/- 0.5) x 10(7) M(-1), respectively. Further adsorption up to 5-6 PGEk molecules on TMO occurrs cooperatively with still high association constant K2(TMO) = (4.0 +/- 1.5) x 10(6) M(-1). TMS oligonucleotide binds the third PGEk molecule rather weakly, K2(TMS) = (8 +/- 2) x 10(5) M(-1). CD spectral analysis revealed that G-quadruplex structure formed by TMO have undergone a partial unfolding by binding of PGEk molecules while single-stranded structure formed by TMS was not affected by binding PGEk. Thus, the tertiary structure of DNA and the number of adsorbed PGEk molecules formed biologically active compounds PGEk: TMO and PGEk: TMS were defined, which are able to penetrate through the membrane of proliferating cells and to suppress their growth.     

High-level expression and purification of human epidermal growth factor with SUMO fusion in Escherichia coli

Human epidermal growth factor (hEGF) can stimulate the division of various cell types and has potential clinical applications. However, the high expression of active hEGF in Escherichia coli has not been successful, as the protein contains three intra-molecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the hEGF gene with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-hEGF fusion gene that was highly expressed in Origami (DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-hEGF, was up to 38.9% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease to obtain the native hEGF, which was further purified by Ni-NTA affinity chromatography. The result of the reverse-phase HPLC showed that the purity of the recombinant cleaved hEGF was greater than 98%. The primary structure of the purified hEGF was confirmed by N-terminal amino acid sequencing and MALDI-TOF mass spectroscopy analysis. Using the method of methylthiazoletetrazolium, the mitogenic activity on Balb/c 3T3 cells of the purified hEGF was comparable to that of commercial hEGF.     

High-level expression, purification, and crystallization of recombinant rat leukotriene C(4) synthase from the yeast Pichia pastoris

Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.     

alpha-Factor directed expression of the human epidermal growth factor in Saccharomyces cerevisiae

Expression kinetics of the human Epidermal Growth Factor (hEGF) from the alpha-factor prepro region in a 2-mum based plasmid was studied in Saccharomyces cerevisiae. Production of hEGF was highly medium de pendent as a chemically defined, nonenriched media had a significantly lower yield than did enriched media. Also cells grown on yeast nitrogen base without amino acids with casamino acids degraded the hEGF after cell growth as opposed to a yeast extract, peptone, and dextrose (YEPD) medium, which elicited no measurable extracellular proteolysis of the hEGF. alpha-factor directed production kinetics of hEGF on the YEPD medium were growth associated, secretion limitations and extracellular degradation were negligible, and the hEGF was nearly 100% selectively secreted. With sufficient agitation, shake flask experiments were representative of aerated controlled batch fermentations. No effect of high cell density was observed on cell growth or hEGF production kinetics. The hollow fiber bioreactor had no direct effect on the substrate or protein yields of S. cerevisiae, however the low oxygen transfer capacity of the membrane was not sufficient to support respiration.     

An improved procedure for production of human epidermal growth factor from recombinant E. coli

An improved procedure for the fermentation and purification of human epidermal growth factor (hEGF) was developed. Recombinant Escherichia coli HB-101 [lacUV5omp08hEGF] harboring plasmid lacUV5omp08hEGF encoding hEGF was used in fermentation to increase levels of hEGF. Medium composition, and the levels of inoculum, inducer (isopropyl-beta-D-thiogalactoside) and ampicillin were optimized with respect to volumetric fermentation of hEGF. As a result, the hEGF concentration reached a high value of 242 mg l(-1) and the amount of heterogeneous protein decreased by 62% compared with that before optimization in batch fermentation. High-quality hEGF was purified from the fermentation culture by centrifugation, salting-out, resuspension, recentrifugation and finally gel chromatography on a Grad-iFrac System using Sephadex G-50 superfine. The purity of hEGF and the total yield were more than 94% and higher than 36%, respectively, and SDS-PAGE of the purified hEGF demonstrated a single band corresponding to an hEGF standard. In particular, a very important phenomenon was found, i.e. that the amount of heterogenous protein in fermentation broths cultured in media with high concentrations of lactose is far less than that cultured in media with high concentrations of glucose.     

Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of <Emphasis Type="Italic">Vibrio mimicus</Emphasis> metalloprotease

Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.     

Secretion of recombinant human epidermal growth factor into the periplasm in Escherichia coli cells]

Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E. coli outer membrane major proteins (OmpF) is controlled by tac promoter. The increase of the hEGF yield was achieved by the multiplication of the gene copies. The hEGF in bacterial cells was secreted into periplasm. The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture. The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein. The preparation proved to be biologically active.     

Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase ysc(alpha) was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.     

Semiquantitative immunoblots of membrane protein-epidermal growth factor

Membrane proteins or cytokines are sometimes difficult to isolate and purify. Our group recently concentrated on epidermal growth factor (EGF) protein expression studies. Mature EGF was initially identified from mouse submaxillary gland extract as a stimulator of eyelid opening and incisor eruption when injected into newborn mice and rats. The EGF precursor is a transmembrane protein with eight additional EGF-like repeats. Our previous study has shown that the EGF precursor without these eight EGF-like repeats (hEGF) was biologically active. Here, we introduce a modified method for rapid detection of hEGF. The membranous protein was directly extracted from various organs of transgenic mice (including the submandibular gland, kidney, liver, heart, and testis) with two different buffers and easily detected by semiquantitative immunoblotting.     

Improved expression of recombinant plant-made hEGF

Key message

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF.

Abstract

Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.     

DNA-binding properties of an adenovirus 289R E1A protein.

An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

A man-made gene coding for [21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The [21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.     

Growth factor engineering by degenerate homoduplex gene family recombination

There is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of "top-strand" oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic "scaffold" oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human-mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.     

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.     

不同流加发酵方式对重组大肠杆菌细胞外Hegf表达水平的影响

人表皮生长因子 (ｈＥＧＦ)是由 5 3个氨基酸组成的单链多肽 ,内有 3个二硫键。它具有多种重要的生物学功能 ,能够促进表皮细胞的生长繁殖 ,加速皮肤和角膜创伤的修复 ,加速胃溃疡的治疗和抑制胃酸的分泌 ,具有广阔的医疗应用前景[1 ] 。最近又发现ｈＥＧＦ在化妆品产业中有潜在的重大应用[2 ] 。这些都促进了人们试图采用基因工程技术大规模地生产ｈＥＧＦ。1982年Ｓｍｉｔｈ等人首先在Ｅ .ｃｏｌｉ中以融合蛋白形式实现了ｈＥＧＦ的表达[3 ] ,随后在枯草杆菌和酵母菌中亦相继实现了表达[4] 。由于ｈＥＧＦ在酵母菌中表达水平很低 ,大肠杆…