Levels Of Recombinant Protein Production Research Articles

Separate-Component-Stabilization System for Protein and Dna Production Without the Use of Antibiotics

Plasmid instability is a significant concern in the industrial utilization of microorganisms for protein or DNA production. Here we report on the development of a new and highly effective stabilization system based on the use of the ccd antidote/poison genes. For the first time, we separated the antidote gene from the poison gene: localizing the former in the plasmid and integrating the latter in the bacterial chromosome. We show that this separate-component-stabilization (SCS) strategy: (i) allows for perfect stabilization without the use of antibiotics; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e., used in most expression systems). Finally, we demonstrate that the SCS system increases by five the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene.

His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors

We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production.

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.

Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy. Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade. In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells. In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls. Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases. Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures. Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs.

A rapid selection/amplification procedure for high-level expression of recombinant protein in a metal-amplifiable mammalian expression system

We describe a strategy for the selection and amplification of foreign gene expression in Chinese hamster ovary (CHO) cells employing a metallothionein gene-containing expression vector. This report describes an amplification procedure that results in an enrichment of clones exhibiting high levels of recombinant protein production and reduces the labour required for screening recombinant cell lines.

33] Expression of green fluorescent protein using baculovirus vectors

The expression of heterologous genes in insect cells using baculovirus vectors is commonly used to achieve a high level of recombinant protein production. The expression system exploits the promoters from a number of hyperexpressed virus genes that are activated in the very late stages of the Autographa californica nucleopolyhedrovirus (AcMNPV) replication cycle. The most widely used of these are the polyhedrin gene (polh) and p10 (p10) gene promoters. Replacement of either the polh or p10 coding region with that of a foreign gene results in the formation of a recombinant virus with the foreign gene under control of the hyperexpressed promoter. Recombinant viruses may be propagated in insect cells cultured in vitro, for example, Spodoptera frugiperda or Trichoplusia ni cells, or may be used to infect insect larvae. In the latter case, recombinant viruses are usually prepared in which the polh and p10 remain intact, so that larvae may be infected by the natural route of feeding upon a polyhedra-containing diet. In this case, the foreign gene is expressed under control of a copy of the polh or p10 promoter, located in a nonessential region of the virus genome, for example, just upstream of the polh promoter.

Low‐Cost Serum‐Free Medium for the BTI‐Tn5B1–4 Insect Cell Line

The BTI-Tn5B1-4 insect cell line, commercially available as the High Five cell line (Invitrogen), supports higher levels of recombinant protein production compared to existing insect cell lines. Proprietary serum-free media such as ExCell 405 (JRH Biosciences), Express Five (Life Technologies), IS BAC (Irvine Scientific), and CCM3 (HyClone) are available which were developed specifically for a suspension culture of High Five cells. While these media are highly optimized, a lower cost alternative is desirable for large-scale protein production which is also serum-free and supports good cell growth (>5 x 10(6) cells/mL) and recombinant protein production (>50 mg/L of secreted protein). The amino acid and carbohydrate metabolism of the Tn5B1-4 cells was first examined. It was found that asparagine was nearly depleted during batch growth in Ex-Cell 405, without limitations in glutamine, other amino acids, or glucose. Alanine also accumulated to about 35 mM during growth. We then extended the formulation techniques for medium development used for Spodoptera cell lines to the Tn5B1-4 cell line. A medium based on IPL-41 basal medium, Hy-Soy protein hydrolysate (Quest, International), yeastolate ultrafiltrate, a lipid-sterol emulsion, and Pluronic F-68 was developed. Dextran sulfate (100 microg/mL) was used to induce a single cell suspension culture. This medium is denoted as ISYL and performs best when supplemented with a 2.5% lipid-Pluronic F-68 mixture. Supplementation with additional aspargine in a 1.5% lipid-Pluronic F-68 mixture did not improve growth, suggesting that a lipid was growth-limiting and not an amino acid. Ex-Cell 405 and ISYL with 2.5% lipid-Pluronic F-68 supplement supported virtually identical growth rates, extent of growth (ca. 6.0 x 10(6) cells/mL) in an 80% oxygen atmosphere, and supported production of SEAP (secreted human alkaline phosphatase) at a volumetric level of about 65-70 mg/L. Thus, the less expensive ISYL medium can deliver acceptable performance and may be suitable for large-scale insect cell cultures.

Adenoviral Vector-Mediated Gene Transfer into Sheep Arteries Using a Double-Balloon Catheter

The potential for catheter-based in vivo delivery of genetic material to the arterial wall is incompletely explored. We evaluated the level of recombinant protein production as well as the anatomic distribution and duration of gene expression following adenoviral vector-mediated gene transfer into sheep arteries via a double balloon catheter. Catheters were positioned in the carotid or femoral arteries of 20 sheep via a combined percutaneous and surgical approach, and virions infused over a 30-min period. Three days later, recombinant gene expression was identified in approximately 30% (range 0-80%) of the luminal endothelial cells within the targeted area of the artery. Persistent recombinant protein expression was identified histochemically for up to 4 weeks, although the number of positive cells decreased steadily. High levels of both beta-galactosidase (beta-Gal) activity and protein (mean 20 mU and 44 ng per vessel) were measured in vessel extracts 3 days after gene transfer, again decreasing significantly over a 4-week period. Transgene expression was limited almost entirely to the intima and adventitia; adventitial gene transfer occurred virtually exclusively along the vasa vasorum. In comparison to previous studies of catheter-based gene transfer, adenoviral vectors delivered by double balloon catheter resulted in a particularly high efficiency of endothelial cell gene transfer. The efficiency and amount of recombinant gene expression achieved in this study suggest that catheter-based gene delivery may eventually be applicable to the treatment of focal human arterial disease.

Strategies in high-level expression of recombinant protein in Escherichia coli.

The accumulation of acetate is one of the most commonly encountered problems in attaining high levels of recombinant protein production using E. coli. Two different approaches are examined to reduce the rate of acetate formation. The effects of reduced acetate accumulation on recombinant protein production were also investigated. In the first approach, E. coli mutant strains deficient in enzymes involved in the acetate synthesis pathways were isolated and characterized. The level of specific production of beta-galactosidase by the mutant strain is three times higher than its parent strain. In another approach, metabolic engineering techniques were employed to fine-tune the central metabolic pathways to reduce the amount of acetate formation. The resulting strain, which carries the acetolactase synthase gene from B. subtilis, is successful in maintaining a very low level of acetate accumulation. The ALS-containing strain is also capable of producing higher levels of recombinant protein than its parent strain.

In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries.

We studied the ability of adenoviral vectors to achieve gene transfer into injured arteries. A recombinant adenoviral vector expressing a nuclear-targeted beta-galactosidase gene was constructed and infused into balloon-injured rat carotid arteries. Three days after gene transfer, recombinant gene expression was assessed quantitatively by (1) measuring beta-galactosidase antigen and activity in tissue extracts and (2) histochemical staining and counting of cells expressing beta-galactosidase. Exposure of injured carotid arteries to increasing concentrations of the vector (10(8) to 10(10) plaque-forming units per milliliter) resulted in a dose-responsive increase in beta-galactosidase expression, with peak expression of approximately 43 mU or 25 ng beta-galactosidase per vessel. Microscopic examination of histochemically stained arteries demonstrated gene transfer limited to the vascular media; transduced cells were identified immunohistochemically as smooth muscle cells. Counting of both histochemically stained and total nuclei in the media revealed that approximately 30% of the cells in the media of the injured vessels were transduced. Calculations based on both counting cells and on the level of beta-galactosidase expression in tissue extracts suggested the presence of 5000 to 10,000 transduced cells per 10 mm of vessel. Arteries infused with either vehicle only, a control adenoviral vector, or liposomes combined with the vector plasmid contained little or no evidence of beta-galactosidase expression. High levels of in vivo beta-galactosidase expression persisted for at least 7 days after gene transfer but declined significantly by day 14. We conclude that adenoviral vector-mediated gene transfer into the injured rat carotid artery results in efficient gene transfer into the vascular media, with levels of recombinant protein production significantly higher than any previously reported in arterial gene transfer studies. Adenoviral vectors appear to be particularly useful agents for in vivo arterial gene transfer.

Schistosoma Mansoni Tropomyosin: Production and Purification of the Recombinant Protein and Studies on its Immunodiagnostic Potential

A cDNA that encodes Schistosoma mansoni tropomyosin, except for 10 amino acids at the amino terminus, has been cloned into a pOTSNCO plasmid vector. Induced expression resulted in a constant level of recombinant protein production. The recombinant S. mansoni tropomyosin was purified from preparative SDS-PAGE gel and by a combination of 20% ammonium sulfate fractionation and fast protein liquid chromatography-ion-exchange chromatography. The purified recombinant S. mansoni tropomyosin was tested as an immunodiagnostic reagent in Western blot and enzyme-linked immunosorbent assays. Sera from individual patients with chronic S. mansoni infection, but not S. haematobium, S. japonicum, parasitic infections other than schistosomiasis, and without infection reacted with the recombinant tropomyosin. The species specificity of S. mansoni tropomyosin suggests that further study of its potential as an immunodiagnostic reagent is warranted.