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Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield.

ABSTRACT: BACKGROUND: Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. RESULTS: We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm) or degraded (cytoplasm) whereas at 18°C, expression was improved especially in the periplasm of C41(DE3) cells and in the cytoplasm of oxidizing ?trx/?gor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3) cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This hypothesis is supported by the fact that use of minimal media could enhance protein production compared to nutrient-rich LB media. CONCLUSIONS: We have found optimal conditions for heterologous expression of U24 from Human Herpesvirus type-6 in E.coli and have demonstrated that milligram quantities of pure protein can be obtained. Strained metabolic conditions such as low temperature, minimal media and an oxidizing environment appeared essential for high-level, full-length protein production and this information may be useful for expressing other membrane proteins of interest.

SUBMITTER: Tait AR

PROVIDER: S-EPMC3155487 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield.

Tait Andrew R AR Straus Suzana K SK

Microbial cell factories 20110629

<h4>Background</h4>Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task ...[more]

PMID: 21714924

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Project description:BackgroundAs a white biotechnological trend, esterases are thought to be among the most active enzymes' classes in biocatalysis and synthesis of industrially importance organic compounds. Esterases are used in many applications such as the manufacture of pharmaceuticals, cosmetics, leather, textile, paper, food, dairy products, detergents, and treatment of some environmental pollutants.ResultsA poly-histidine moiety was added to the C-terminal end of the Geobacillus sp. gene encoding carboxyl esterase (EstB, ac: KJ735452) to facilitate one-step purification. This recombinant protein was successfully expressed in Escherichia coli (E. coli) under control of Lambda promoter (λ). An open reading frame (ORF) of 1500 bps encoding a polypeptide of 499 amino acid residues and a calculated molecular weight (54.7 kD) was identified as carboxyl-esterase B due to its conserved glycine-X-serine-X-glycine motif (G-X-S-X-G) and its high similarity toward other carboxyl esterases, where the 3-D tertiary structure of EstB was determined based on high homology % (94.8) to Est55. The expression was scaled up using 7-L stirred tank bioreactor, where a maximum yield of enzyme was obtained after 3.5 h with SEA 51.76 U/mg protein. The expressed protein was purified until unity using immobilized metal affinity chromatography (IMAC) charged with cobalt and then characterized. The purified enzyme was most active at pH 8.0 and remarkably stable at pH (8-10). Temperature optimum was recorded at 65 °C, and it kept 70% of its activity after 1-h exposure to 60 °C. The active half-live of enzyme was 25 min at 70 °C and a calculated T melting (Tm) at 70 °C. The determined reaction kinetics Michaelis-Menten constant (Km), maximum velocity rate (Vmax), the turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the pure enzyme were found 22.756 mM, 164.47 U/ml (59.6 min-1), and (2.619 mol/ min), respectively.ConclusionCreation of a recombinant 6 × -His estB derived from a thermophile Geobacillus sp. was performed successfully and then overexpressed under λ-promoter. In a bench scale bioreactor, the overexpression was grown up, followed by one-step purification and biochemical characterization. The recorded promising pH and temperature stability properties suggest that this expressed carboxyl esterase could be used in many industrial sectors.

Project description:Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A, a small, bispecific molecule with affinity for both HSA and the novel target was identified. The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination.

Dataset Information

Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield.

Publications

Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield.

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