Solubilization of Recombinant Ovine Growth Hormone with Retention of Native-like Secondary Structure and Its Refolding from the Inclusion Bodies of Escherichia coli

Khan, Rizwan Hasan; Rao, K.B.C. Appa; Eshwari, A. N. S.; Totey, S.M.; Panda, Amulya K.

doi:10.1021/bp980071q

Cited by 98 publications

(67 citation statements)

References 35 publications

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“…Inclusion bodies purified as described above were resuspended in IB denaturation buffer (100 mM Tris [pH 12.0], 2 M urea), conditions that unfolded the proteins only partially, allowing effective renaturation (35). To avoid precipitation of the Mod proteins, the concentration of the proteins was adjusted to 1 mg/ml and the sample was transferred to a dialysis bag (type 20 dialysis membrane; Biomol, Hamburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

Tiemann

Depping

Gineikiene³

et al. 2004

J Bacteriol

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Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Altand ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.Posttranslational ADP-ribosylation of proteins is catalyzed by ADP-ribosyltransferases (ADP-RTs), which have been identified in viral, bacterial, and eukaryotic systems. Transfer of the ADP-ribose moiety from the substrate NAD ϩ to a specific amino acid residue, frequently histidine or arginine within a target protein, modulates the activity of the acceptor. ADP-ribosylation changes the electrostatic potential of a target protein by introducing two phosphate groups and may affect protein-DNA as well as protein-protein interactions. ADP-RTs were initially discovered as the exotoxins of pathogenic bacteria. Therefore, most of our knowledge concerning these proteins has been gained by biochemical, genetic, and structural studies performed on bacterial toxins, such as those produced by Corynebacterium diphtheriae (15, 30), Bordetella pertussis (34), Vibrio cholerae (46), Pseudomonas aeruginosa (33), and Escherichia coli (45). New putative bacterial toxins were found to be encoded in the genomes of Streptococcus pyogenes and Salmonella enterica serovar Typhi (50).To date, the family of ADP-RTs comprises more than 40 enzymes, including bacterial exotoxins as well as a variety of other enzymes, such as the eukaryotic mono-and poly-ADPRTs, which include T-cell differentiation alloantigens like RT6 (9), poly-ADP-RTs (56), the dinitrogenase reductase regulation factor DraT (51,77), and enzymes encoded by T-even bacteriophages. The bacteriophage T4 gene products Alt (76 kDa), ModA (23 kDa), and ModB (24 kDa) appear to actively regulate gene expression during the transition from host t...

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Section: Methodsmentioning

confidence: 99%

ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

Tiemann

Depping

Gineikiene³

et al. 2004

J Bacteriol

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“…[21][22][23] It has also been found that the solubilization of r-hGH inclusion bodies at alkaline pH in the presence of 2 M urea solution was more efficient than at alkaline pH only. This method resulted in about 94% of solubility, and the overall yield of the purified r-hGH was about 50% of the initial inclusion body proteins.…”

Section: Solubilization Of R-hghmentioning

confidence: 97%

Improved Solubilization of Recombinant Human Growth Hormone Inclusion Body Produced inEscherichia coli

Sonoda¹,

Sugimura²

2008

Bioscience, Biotechnology, and Biochemistry

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Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGHdependent cell line.

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“…DISCUSSION We used size-exclusion chromatography to fold the ␤ 1b -subunit from voltage-activated calcium channels into active monomers. The ␤ 1b -subunit with 65 kDa is the largest protein so far refolded by this method that was initially applied to fold smaller proteins with molecular masses between 14 -20 kDa (17,22,23). The successful use of this method for the ␤ 1b -subunit might be associated to its multi-domain structure (11) and, whether the SEC-based refolding procedure can be generally applied to larger proteins lacking such a feature, can not be anticipated from this work.…”

Section: Effect Of the Folded-␤ 1b On ␣ 1c -Calcium Channels Expressementioning

confidence: 98%

Folding of Active Calcium Channel β1b -Subunit by Size-exclusion Chromatography and Its Role on Channel Function

Neely

Garcia‐Olivares

Voswinkel

et al. 2004

Journal of Biological Chemistry

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Voltage-gated calcium channels mediate the influx of Ca 2؉ ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming ␣ 1 -subunit and the auxiliary ␤-and ␣ 2 ␦-subunits. The ␤-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four ␤-subunit genes have been cloned, ␤ 1-4 , with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The ␤ 1b -subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming ␣ 1 -and the regulatory ␤-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the ␤ 1b -subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The ␤ 1b -subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the ␣ 1 -subunit (the ␣-interaction domain). Using the cutopen oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac ␣ 1 -subunit (␣ 1C ) co-injected with folded-␤ 1b -protein or ␤ 1b -cRNA. We demonstrate that the co-expression of the ␣ 1C -subunit with either folded-␤ 1b -protein or ␤ 1b -cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the ␤ 1b -subunit primarily modulates channel activity, rather than expression.Changes in the intracellular calcium concentration regulate a variety of cellular functions such as neurotransmission, muscle contraction, hormone secretion, and gene expression. High threshold voltage-activated calcium channels are the main route for calcium entry in electrically excitable cells. They are membrane protein complexes composed of at least three nonhomologous subunits, the ␣ 1 -, ␤-, and the ␣ 2 /␦-subunit. Through molecular cloning, at least 10 genes encoding mammalian ␣ 1 -subunits (␣ 1A-I and ␣ 1S ) have been identified in different cell types (1). Although the ␣ 1 -subunit encompasses all the structural elements of a functional voltage-activated calcium channel, such as the ion-conduction pathway, the voltage sensor, and drug-binding sites, the ␤-subunit seems to be essential for channel performance (2) and to be acting at two levels: (i) channel expression by interfering with the ␣ 1 -subunit endoplasmic reticulum (ER) 1 -retention signal to facilitate intracellular trafficking (3, 4), and (ii) channel activity by modifying the electrophysiological properties of the channel (5-7).Two highly conserved sequences have been identified as the primary interaction site between the ␣ 1 -and the ␤-subunit, the ␣ 1 -subunit interaction domain (AID) that lies within the c...

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Solubilization of Recombinant Ovine Growth Hormone with Retention of Native-like Secondary Structure and Its Refolding from the Inclusion Bodies of Escherichia coli

Cited by 98 publications

References 35 publications

ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

ModA and ModB, Two ADP-Ribosyltransferases Encoded by Bacteriophage T4: Catalytic Properties and Mutation Analysis

Improved Solubilization of Recombinant Human Growth Hormone Inclusion Body Produced inEscherichia coli

Folding of Active Calcium Channel β1b -Subunit by Size-exclusion Chromatography and Its Role on Channel Function

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