Bacterial expression and refolding of human trypsinogen

Hohenblum, Hubertus; Vorauer-Uhl, Karola; Katinger, Hermann; Mattanovich, Diethard

doi:10.1016/j.jbiotec.2003.10.022

Cited by 25 publications

(14 citation statements)

References 27 publications

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“…Otherwise, despite the benefits of recombinant enzyme production, the trypsin expression in E. coli heterologous systems has also shown a common self-digestion enzyme and a multiple disulfide bound formation, due to the reducing environment in E. coli bacteria cytoplasm (Verheyden et al, 2000). Novel strategies have been applied to solving these difficulties, which included periplasmic expression, a known oxidant environment (Vasquez et al, 1989;Fukuoka et al, 2002), the use of a thioredoxin redutase deficient mutant strain (Verheyden et al, 2000;Prinz et al, 1997), and cytosolic expression followed by the use of oxidating buffers, as developed in this work (Shan et al, 2003;Hohenblum et al, 2004). The enzyme was mainly active at pH 8.0, but residual activity at pH 7.0 and 9.0 was recorded (Fig.…”

Section: Discussionmentioning

confidence: 99%

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Magalhães

Fragoso

Souza

et al. 2007

Arch Insect Biochem Physiol

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The Mexican bean weevil, Zabrotes subfasciatus, feeds on several seeds such as Vigna unguiculata, Phaseolus vulgaris, and Pisum sativum, causing severe crop losses. This ability to obtain essential compounds from different diets could possibly be explained due to a wide variability of digestive proteinases present in the weevil's midgut. These may improve digestion of many different dietary proteins. Coleopteran serine-like proteinases have not been thoroughly characterized at the molecular level. In this report, a full-length cDNA encoding a trypsin-like protein, named ZsTRYP, was isolated from Z. subfasciatus larvae using RT-PCR, 5' and 3' RACE techniques. The quantitative real-time PCR analysis strongly correlated the Zstryp transcript accumulation to the major feeding developmental larval stage. Zstryp cDNA was subcloned into pET101 vector and expressed in a Escherichia coli BL21(DE3) strain. Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to purify a 29.0-kDa recombinant enzyme. The purified ZsTRYP was then assayed with several synthetic peptide substrates and also challenged with different inhibitors. The biochemical data allowed us to classify ZsTRYP as a trypsin. Moreover, homology modeling analysis indicated a typical trypsin structural core and a conserved catalytic triad (His(41), Asp(86), and Ser(182)).

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Section: Discussionmentioning

confidence: 99%

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Magalhães

Fragoso

Souza

et al. 2007

Arch Insect Biochem Physiol

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“…Production of recombinant trypsinogen has been attempted mainly using Escherichia coli as a host, however, the low solubility of the produced trypsinogen, with formation of inclusion bodies, requires additional solubilization and refolding steps. 5,6 The methylotrophic yeast Pichia pastoris has been developed as a host for the efficient production and secretion of foreign proteins. 7 Generally, this expression system uses the methanolinducible alcohol oxidase 1 (AOX1) promoter for high cell density cultures with methanol-regulated expression cassettes.…”

Section: Introductionmentioning

confidence: 99%

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Guerrero-Olazarán

Escamilla‐Treviño

Castillo-Galván

et al. 2009

Biotechnology Progress

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Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.

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“…The trypsinogen quantification was performed as described before (Hohenblum et al, 2004b) with a trypsin kinetic assay using p-tosyl-L-arginine methyl ester (TAME) after activation with bovine enterokinase.…”

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confidence: 99%

Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

Baumann

Maurer

Dragosits

et al. 2007

Biotech & Bioengineering

121

100

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High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.5-fold increase of the specific productivity q(P) at low oxygen supply was observed. At the same time the biomass decreased and ethanol was produced, indicating a shift from oxidative to oxidofermentative conditions. Based on these results we designed a feedback control for enhanced productivity in fed batch processes, where the concentration of ethanol in the culture was kept constant at approximately 1.0% (vv(-1)) by a regulated addition of feed medium. This strategy was tested successfully with three different protein producing strains, leading to a three- to sixfold increase of the q(P) and threefold reduced fed batch times. Taken together the volumetric productivity Q(P) increased 2.3-fold.

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Bacterial expression and refolding of human trypsinogen

Cited by 25 publications

References 27 publications

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Molecular and structural characterization of a trypsin highly expressed in larval stage of Zabrotes subfasciatus

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

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