<i>Escherichia coli</i>Expression and Processing of Human Interleukin-1β Fused to Signal Peptides

Curry, Kimberly A.; Yem, Anthony W.; Deibel, Martin R.; Hatzenbuhler, Nicole T.; Hoogerheide, John G.; Tomich, Che-Shen C.

doi:10.1089/dna.1990.9.167

Cited by 11 publications

(15 citation statements)

References 51 publications

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“…The OmpA delivery system has been successfully demonstrated in a number of studies, e.g., studies using streptokinase (18) and TolAII (35). In addition, incomplete cleavage of the PhoA leader peptide was used to produce human interleukin-1␤ (9,11). This can affect the acquisition of the mature and active form of the heterologous protein.…”

Section: Discussionmentioning

confidence: 99%

Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

Manosroi¹,

Tayapiwatana

Götz

et al. 2001

Appl Environ Microbiol

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The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of ⌽M13 and linked to the OmpA signal sequence. The resulting gene, rK2S-gpIII, was inducibly expressed in Escherichia coli XL-1 Blue. The protein was presented on the phage particle. To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-K2S, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-␤-Dthiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/g. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacteriumbased tPA production systems.

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Section: Discussionmentioning

confidence: 99%

Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

Manosroi¹,

Tayapiwatana

Götz

et al. 2001

Appl Environ Microbiol

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“…Hatched box indicates tat promoter and dotted lines indicate LTB signal sequence. and ompA signal sequence, gave only 20% and 50% of processed protein respectively [9]. The use of neutral protease gene signal sequence also showed partial processing of hGH in Bacillus [14].…”

Section: Analysis Of the Expression Of Hgh By Pgghgh-ementioning

confidence: 99%

“…The strategy to use the signal peptide to make the protein enter the secretary pathway has been successful; however, it also has encountered some problems. Most commonly, the processing of the pre- [3,9]. Alternate hosts like Pseudomanas, Bacillus subtilis have also been tried and even these systems do have similar problems associated with it.…”

Section: Introductionmentioning

confidence: 99%

Efficient processing and export of human growth hormone by heat labile enterotoxin chain B signal sequence

Ghorpade

Garg

1993

FEBS Letters

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The heat-labile enterotoxin chain B (LTB) signal sequence was used for the processing and export of human growth hormone (hGH). The protein was completely processed and exported across the cell membrane to accumulate in the periplasmic space in Eschrrichia co/i. The human growth hormone cDNA was cloned as a PCR amplified fragment under the control of tat promoter and translationally fused to the LTB signal sequence. The rate of processing of hGH under the control of the LTB signal sequence was equal to or more than the rate of induction of expression, indicating effictent processmg. The receptor bindmg activity of the processed periplasmtc protein was established in a radio receptor assay.

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“…Sonication used a Labsonic (B Braun) device to provide continuous application of acoustic waves with a variable power input (up to 300 W) and a frequency of 20 kHz, for 1±2 min, until the cell suspension (80 cm 3 ) reached a maximum temperature of 20°C. The sonication was then interrupted and the cell suspension cooled to the initial temperature (10°C) followed by a new sonication cycle.…”

Section: Sonicationmentioning

confidence: 99%

“…The cell suspension (160 cm 3 ) was pumped, at a¯ow rate dependent on pressure homogenization, through the homogenization chamber. Pressure was controlled between 0 and 101.…”

Section: Homogenizationmentioning

confidence: 99%

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

Fonseca

2002

J of Chemical Tech & Biotech

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The release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high-pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low speci®c activities, 0.1±0.3 IU(mg prot) À1. Intracellular total soluble protein release was quanti®ed and modelled by a ®rst order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with speci®c enzyme activities of about 4 IU(mg prot)À1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis.

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Escherichia coliExpression and Processing of Human Interleukin-1β Fused to Signal Peptides

Cited by 11 publications

References 51 publications

Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in Escherichia coli

Efficient processing and export of human growth hormone by heat labile enterotoxin chain B signal sequence

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

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