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.     

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.     

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.     

Rapid purification and partial characterization of recombinant human epidermal growth factor produced by Escherichia coli

Human recombinant EGF, secreted into the extracellular medium by E. coli cells, was purified by a combination of solid phase extraction and HPLC. Using these techniques, the peptide was purified 122-fold, with a recovery of greater than 75%. The purified hEGF manifested no contaminating protein bands on electrophoretic gels. Amino acid analysis of the purified peptide was identical to that of authentic hEGF.     

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.     

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.     

Improvement of hEGF production with enhanced cell division ability using dissolved oxygen responses to pulse addition of tryptone

Tryptone has multiple and complex effects on cell physiology and process performance in pulse fed-batch cultivation of recombinant Escherichia coli. By applying feedback control of dissolved oxygen signal responding to pulse in the feed rate, the production of acetate was avoided and the optimization of production of recombinant human epidermal growth factor (hEGF) was successfully achieved. With the addition of an optimum amount of tryptone along with glucose in the pulse fedbatch cultivation of E. coli, the ability of the cell to divide and the stability of the plasmid within the bacteria were improved. Consequently, segregation of the cells into a viable but non-culturable physiological state was alleviated. Addition of tryptone also enhanced cell respiration before and after hEGF expression and thus further benefited the production of recombinant hEGF. Excessive addition of tryptone resulted in low sensitivity of the oscillation of dissolved oxygen signal and poor operability of pulse fed-batch cultivation as this led to an accumulation of acetate, which weakened the dissolved oxygen signal responses. Consequently, the production of recombinant protein was considerably reduced. By combining the process performance and the positive effect of complex media pulse addition on bacterial metabolism, the optimal production conditions of hEGF were successfully determined. A high cell density of 91 g/L dry cell weight was obtained under these optimal production conditions. Furthermore, a high level of 0.24 g/L hEGF was attained leading to a 32.6% increase in product yield as compared to the controls.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Regulation of hamster embryo development in vitro by carbon dioxide

We have examined the effect of collecting and culturing hamster eight-cell embryos in media containing high levels of bicarbonate and/or CO2 on development in vitro. An approximate doubling in the percentage of embryos developing to the blastocyst stage was observed upon raising the concentration of CO2 in the gas phase from 5% to 10% CO2. Development to the blastocyst stage was not affected by the bicarbonate concentration (6-50 mM), nor by the pH of the medium (6.5-7.4). However, escape of embryos from their zonae pellucidae was pH-dependent (optimum pH 7.1-7.4). We hypothesized that the beneficial effect of high concentrations of CO2 on blastocyst development was due to the action of CO2 as a weak acid in regulating intracellular pH (pHi). To test this hypothesis, eight-cell embryos were cultured under 5% CO2 in media containing various concentrations of organic weak acids (lactic or acetic acids, or the non-metabolizable compound 2,4-dimethyloxazolidine-dione). Embryos cultured in standard medium (TALP) under 5% and 10% CO2 served as low and high controls, respectively. At optimum concentrations, all of the media containing weak acids supported embryo development significantly better than 5% CO2-equilibrated low control medium, and gave a response similar to that obtained with high control medium equilibrated in 10% CO2. These studies demonstrate that culture in a 10% CO2 environment has a marked stimulatory effect on in vitro development of hamster eight-cell embryos and suggest that this effect is due to maintenance of pHi.     

内皮细胞抑制素酵母工程菌的高密度发酵及产物纯化和活性分析

用 30升发酵罐研究了内皮细胞抑制素 (EDN)酵母工程菌P .pastorisGS115 (pPIC9K EDN)的高密度发酵工艺 ,摸索出发酵培养基 ,pH ,诱导时间等对菌体生长和基因表达的影响 .根据所确定的最适条件进行发酵 ,通过补料和甲醇诱导 4 8h后 ,工程菌GS115 (pPIC9K EDN)生物量的A60 0 值达到 2 5 0 ,分泌量为 15 0mg L .发酵液经StreamlineSP ,SepharoseSPFF离子交换柱和Sepharose HeparinHiTrap亲和柱纯化 ,产物纯度达 97%以上 ,回收率为 6 0 % .Western印迹结果表明 ,酵母工程菌GS115 (pPIC9K EDN)表达的重组人内皮细胞抑制素具有天然EDN的免疫原性 .MTT法和抑癌实验结果显示 :纯化产物能抑制人脐静脉内皮细胞的增殖并能抑制移植于小鼠的黑色素瘤的生长 ,其平均抑瘤率是 94 2 % .     

Effect of polyphenol content on the hydrolysis and fermentation of grain sorghum starch

The high polyphenol content of birdproff grain sorghum has been associated with impaired nutritional quality of the grain and with reduced brewing value of birdproof grain sorghum malt due to enzyme inhibition. In this investigation, high polyphenol grain sorghum was evaluated as a feedstock for fermentation ethanol production using NaOH pretreatment to inactivate the polyphenolic compounds prior to hydrolysis with commercial amylases. The polyphenolic inhibition of starch hydrolysis was minimal at a grain sorghum slurry concentration of 20% dry solids, but became pronounced at slurry concentrations of 28% and higher. At these high slurry concentrations the liquefaction and subsequent saccharification and fermentation were markedly improved by alkaline pretreatment. The highest ethanol concentration (12·3%, vol/vol), coupled with the best starch conversion efficiency to ethanol (83·5%), was obtained with a 28% grain sorghum slurry using a partial simultaneous saccharification and fermentation procedure. The residual fermented solids had a crude protein content of 45·4%. Tannic acid decreased yeast cell viability in synthetic media, but had no effect on the hydrolysis or fermentation of grain sorghum starch.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

产纳豆激酶菌株Bacillus sp.ZLK08的分离及酶纯化

获得稳定产纳豆激酶菌株,为高酶活纳豆激酶产生菌的改造奠定基础.取各来源纳豆,用平板梯度稀释法分离菌株,测定菌株酶活,利用16S rDNA鉴定产酶菌株,凝胶过滤法纯化纳豆激酶并SDS-PAGE检测分析.成功获得稳定产酶芽胞杆菌Bacillus sp.ZLK08,16S rDNA分析表明其与GenBank中序列同源率达到99％,液体发酵表明酶活达到2.5 FU/mL,经纯化,SDS-PAGE表明纳豆激酶分子量为28.46 ku.分离出的Bacillus sp.ZLK08菌株能稳定产纳豆激酶且具有较高酶活.     

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.     

Protein transduction domain-hA20 fusion protein protects endothelial cells against high glucose-induced injury

We constructed a plasmid containing a protein transduction domain (PTD) and a human A20 (hA20) gene fragment; the fusion protein was obtained by highly expressing this plasmid in the yeast Pichia pastoris GS115. The plasmid was obtained by adding 9xArg and EcoR? recognition sites to the end of the primer, and 6xHis-Tag and Not? recognition sites to its end. After sequencing, the hA20 gene fragment was inserted into plasmid pPIC9k to construct expression vector pPIC9k-PTD-hA20; then, we transfected GS115 with the vector and induced PTD-hA20 protein expression. We purified protein from the yeast fermentation supernatant using a nickel column. Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose medium (30 mM glucose) and in high glucose medium containing different concentrations of protein. Apoptosis of HUVECs was assayed by TUNEL 72 h later. The biological activity tests indicated that the fusion protein not only passed through the cell membrane freely, but also inhibited apoptosis of HUVECs induced by high glucose levels. We conclude that the fusion protein PTD-hA20 has potential for clinical use.     

The 24kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning,in vivo expression and import pathway of a protein with unusual properties

The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.Abbreviations CNBr cyanogen bromide - IP isoelectric point - NCS N-chlorosuccinimide - NTA nitrilotriacetic acid - omp24 outer envelope membrane protein of spinach chloroplasts - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SV8 protease Staphylococcus aureus V8 protease (Endoprotease Glu-C) - TPT chloroplast triose phosphate/phosphate translocator