Purification and on-column refolding of EGFP overexpressed as inclusion bodies in Escherichia coli with expanded bed anion exchange chromatography.

The enhanced green fluorescent protein (EGFP) was over-expressed in Escherichia coli as inclusion bodies to increase its quantity and to facilitate its purification. Insoluble EGFP has been purified on Q Hyper Z matrix by expanded bed adsorption after solubilization in 8 M urea. The adsorption was made in expanded bed mode to avoid centrifugation. EBA-column refolding was done by elimination of urea and elution with NaCl. The EGFP was obtained as a highly purified soluble form with similar behavior in fluorescence and electrophoresis as native EGFP.

Evaluation of three expanded bed adsorption anion exchange matrices with the aid of recombinant enhanced green fluorescent protein overexpressed in Escherichia coli.

Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE, Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an ultrasonic homogenate of Escherichia coli. Two pH of buffer were tested. Capture was done in an expanded mode whereas elution was done in a packed mode. The same conditions were chosen for evaluation of the three matrices. We observed a loss of EGFP (8-15%) in the through flow fraction especially with the Streamline Q XL matrix, probably due to an aggregation of beads during sample application. The beads of this matrix possess tentacles which probably retain a lot of cellular and molecular debris. The two other matrices gave a good purification of the EGFP (7-15-fold) but the Q Hyper Z matrix appeared to give the best results. It is composed of little size and density beads which lead to a higher exchange surface and then a better mass transfer.

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins.

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography.

Cloning, expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli.

In this report, we describe a two-step chromatographic procedure for the purification of His-tag EGFP by immobilized metal affinity expanded bed adsorption (IMAEBA) as the capture step and size exclusion chromatography as the polishing step. The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists in purifying recombinant proteins. The procedure described allowed the obtention of 230 mg pure EGFP from 1 l simple batch culture with a recovery of 90%.

On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption.

In this report, we describe a new process for the on-line purification of His-tag EGFP (enhanced green fluorescent protein) taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption (IMAEBA). The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists interested in purifying recombinant proteins. After evaluating the different flow-rates and ultrasonic probe sizes, the on-line purification was tested. After ultrasonic treatment, 70% of the cells were broken and 90% of free EGFP was recovered after IMAEBA. In our conditions, more than 450 mg of EGFP were obtained in 15 h. On-line bioreactor-ultrasonic probe-immobilized metal affinity expanded bed adsorption is a rapid automated technique for obtaining large quantities of pure EGFP.

Acyl-CoA elongase expression during seed development in Brassica napus.

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.

Enzymic activities and gene expression of enzymes of the acyl-CoA elongase during rapeseed development.

Enzymic activities and gene expression of oleoyl-CoA elongase were studied during seed development using two different rapeseed cultivars, high-erucic-acid rapeseed (HEAR) and low-erucic-acid rapeseed (LEAR). The overall elongase activities were maximal in HEAR between the fourth and eighth weeks after pollination (WAP) and absent in LEAR. The 3-ketoacyl-CoA synthase (condensing enzyme, CE) mRNA levels and the developmental profiles in the two cultivars were different since maximal expression levels were detected in HEAR and LEAR at WAP 4 and WAP 6, respectively. Anti-CE antibodies revealed two proteins of 60 and 67 kDa in both cultivars and an additional reacting protein of 57 kDa in HEAR.

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.

Purification and activity of a wheat 9-kDa lipid transfer protein expressed in Escherichia coli as a fusion with the maltose binding protein.

The cDNA encoding a wheat (Triticum durum) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage. Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion. However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.

[Expression of recombinant human hemoglobin in plants]. / Expression d'hémoglobine humaine recombinante dans les plantes.

Human utilization of recombinant proteins of therapeutical interest, as hemoglobin, implies that the transgenic host allows a low cost production of the active proteins with minimal risks of pathogen contamination. In this regard, the use of transgenic plants could be of great interest. In particular, the systems based on plants could be one of the most economical transgenic system, compared with the others, because biomass obtention in fields is not expensive.