High-level production and one-step purification of biologically active human growth hormone in Escherichia coli

Mukhija, Reema; Rupa, Prithy; Pillai, Devika; Garg, Lalit C.

doi:10.1016/0378-1119(95)00525-b

Cited by 45 publications

(21 citation statements)

References 18 publications

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“…The protein folds into a four-helix bundle structure with two disulfide bridges, one connecting distant parts of the molecule involving amino acid residues 53 and 165 (large loop) and another between residues 182 and 189 (small loop) (19). The large-scale requirement of r-hGH necessitates its high-level expression in E. coli as inclusion bodies (20). However, expression of the protein along with fusion tag and subsequent use of high concentrations of chaotropic reagents for solubilization and purification makes the overall process more complex and expensive as the yield of bioactive r-hGH is lowered (21).…”

Section: High-level Expression Of Recombinant Proteins Inmentioning

confidence: 99%

See 1 more Smart Citation

Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli

Patra

Mukhopadhyay

Mukhija

et al. 2000

Protein Expression and Purification

209

142

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Section: High-level Expression Of Recombinant Proteins Inmentioning

confidence: 99%

“…To clone hGH without any tag and its signal sequence, the cDNA fragment coding for hGH was excised with HinfI-HindIII from pRMhGH (20). This excised cDNA fragment was lacking the first 18 bp.…”

Section: Cloning and Expression Of R-hghmentioning

confidence: 99%

Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli

Patra

Mukhopadhyay

Mukhija

et al. 2000

Protein Expression and Purification

209

142

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“…Human growth hormone (hGH) consists of 191 amino acid residues folded into a four-helix bundle structure with two disulfide bridges, with a wide range of biological functions as protein synthesis, cell proliferation and metabolism [1,2]. Recombinant human growth hormone (rhGH) is a type of GH produced by recombinant DNA technology identical to human growth hormone [3,4].…”

Section: Introductionmentioning

confidence: 99%

Determination of IPTG in Recombinant Human Growth Hormone with Ion Chromatography and Pulsed Electrochemical Detection

Wang¹,

Lin²

2017

Mod Chem Appl

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A novel method for the determination of isopropyl-β-D-thiogalactopyranoside (IPTG), a sulfur-containing compound, a pharmaceutical additive in commercial recombinant human growth hormone (rhGH), has been developed using pulsed electrochemical detection (PED), following ion Chromatographic separation. A Dionex-500 ion chromatograph coupled with an electrochemical detector was employed, equipped with a gold working electrode and an Acclaim 300 analytical C18 column, with a mobile phase consisting of sodium acetate (NaOAc) buffer (pH 5.45, 0.01 mol/L) and acetonitrile (ACN)(90/10, v/v). Upon optimization, IPTG was found to have a limit of detection of 1 ng/mL (0.1 μmol) with 25 uL injection volume. This method was successfully applied to the determination of IPTG in rhGH samples with the characteristics of simplicity, high sensitivity and good repeatability.

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“…Expression of the recombinant LTB and preparation of different cellular fractions were carried out as described earlier (50). E. coli cells harboring mutant or WT plasmids were grown in 50 ml of LB medium in the presence of ampicillin (50 g/ml) at 37°C in a gyratory shaker until A 600 reached 0.4.…”

Section: Expression Of Mutant Ltb Gene and Preparation Of Cellular Frmentioning

confidence: 99%

Deletion mutations in N-terminal α1 helix render heat labile enterotoxin B subunit susceptible to degradation

Alone¹,

Malik²,

Krishnan³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal ␣1 helix lies outside the core structure. However, the function of the N-terminal ␣1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the ␣1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the ␣1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the ␣1 helix also resulted in loss of expression. However, a single amino acid deletion outside the ␣1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcriptiontranslation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants.gene expression ͉ site-directed mutagenesis ͉ coupled transcription-translation D iarrhea caused by the enterotoxigenic Escherichia coli (ETEC) is a major cause of death in developing countries, especially among children, with an estimated mortality of 1.5 million cases every year (1-3; for review, see ref. 4). Approximately 20% of cases of traveler's diarrhea are caused by ETEC, and thus the organism spreads to the developed countries (4, 5). ETEC produces a number of virulence factors, such as enterotoxins and colonization factors. Of these, the heat-labile (LT) and heat-stable enterotoxins produced by the ETEC are the major virulence factors responsible for its pathogenicity (5, 6). The LT belongs to a family of bacterial proteins designated heat-labile enterotoxins and shares phenotypic and genotypic similarities with other members of the family such as cholera toxin produced by Vibrio cholerae (7, 8; for review, see ref. 9).The mature toxin consists of a single A polypeptide (LTA) and five B polypeptides (LTB) (10-12). LTB and cholera toxin B show a high degree of homology, with 85% conservation of amino acids (13). The genes of the two LT subunits, eltA and eltB, are transcribed as a single polycistronic mRNA (14) and are expressed with signal peptides. The two subunits are synthesized as a precursor protein, and each subunit has its own ribosome-binding site (15-17). After cleavage of the signal peptide, the two subunits of LT are released into the periplasmic space where they spontaneously assemble into a mature holotoxin (18,19). LTA is known to influence LTB oligomeriz...

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High-level production and one-step purification of biologically active human growth hormone in Escherichia coli

Cited by 45 publications

References 18 publications

Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli

Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli

Determination of IPTG in Recombinant Human Growth Hormone with Ion Chromatography and Pulsed Electrochemical Detection

Deletion mutations in N-terminal α1 helix render heat labile enterotoxin B subunit susceptible to degradation

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