Soluble expression and purification of porcine pepsinogen from Pichia pastoris

Yoshimasu, Mark A.; Ahn, Jong-Kun; Tanaka, Takuji; Yada, Rickey Y.

doi:10.1016/s1046-5928(02)00003-1

Cited by 32 publications

(9 citation statements)

References 32 publications

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“…11 ). However, at the similar pH, the K m and k cat / K m values for protein were 40 ± 1 μM and 2640 ± 40 s −1 mM −1 21 . It is apparent that the catalysis efficiency of pepsin on NA is approximately 10,000 times lower than that on protein.…”

Section: Discussionmentioning

confidence: 88%

Digestion of Nucleic Acids Starts in the Stomach

Liu

Zhang

Dong

et al. 2015

Sci Rep

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The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3′-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology.

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Section: Discussionmentioning

confidence: 88%

Digestion of Nucleic Acids Starts in the Stomach

Liu

Zhang

Dong

et al. 2015

Sci Rep

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“…26 The SUC2 signal peptide has also been used for secretion studies with -1-antitrypsin but only 20% of the protein was secreted. 107 The acid phosphatase (PHO) 108 signal peptide has also been successfully used to express mouse 5-HT5A serotonin receptor 109 and porcine pepsinogen, 110 however, other proteins expressed and secreted using PHO have had incorrect amino-terminal processing. 111 The pHIL-S1 vector contains the acid phosphatase signal and was used to express human midkine.…”

Section: Use Of the Signal Sequencementioning

confidence: 99%

“…One form of porcine pepsinogen expressed in P. pastoris that was thought to contain O-linked glycosylation was found to be more thermostable than the corresponding unglycosylated forms as determined by both the functionality of the heat-treated samples as well as by analysis of the melting point transitions using circular dichroism. 110 Functional analysis of the activin receptor type IIA extracellular domain in both its fully glycosylated and de-glycosylated forms showed only minor differences in ligand-binding ability; however, the de-glycosylated form was active for a significantly shorter period of time and was less resistant to the chemical regeneration conditions that were employed. 143 Glycosylation can also improve stability of proteins that are not normally glycosylated in their native state.…”

Section: Thermostabilitymentioning

confidence: 99%

Expression of heterologous proteins inPichia pastoris: a useful experimental tool in protein engineering and production

2005

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The use of the methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of recombinant proteins has become increasing popular in recent times. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P. pastoris is also a eukaryote, and thereby provides the potential for producing soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality. Additionally, linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures, or alternatively the target protein and its cognate binding partners, to be expressed. A further benefit of the P. pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest, thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems. The purpose of this review is to summarize important developments and features of this expression system and, in particular, to examine from an experimental perspective the genetic engineering, protein chemical and molecular design considerations that have to be taken into account for the successful expression of the target recombinant protein. Included in these considerations are the influences of P. pastoris strain selection; the choice of expression vectors and promoters; procedures for the transformation and integration of the vectors into the P. pastoris genome; the consequences of rare codon usage and truncated transcripts; and techniques employed to achieve multi-copy integration numbers. The impact of the alcohol oxidase (AOX) pathways in terms of the mut+ and mut(s) phenotypes, intracellular expression and folding pathways is examined. The roles of pre-pro signal sequences such as the alpha mating factor (alpha-MF) and the Glu-Ala repeats at the kex2p cleavage site on the processing of the protein translate(s) have also been considered. Protocols for the generation of protein variants and mutants for screening for orphan cognate binding partners and the use of experimental platforms addressing the molecular recognition behaviour of recombinant proteins such as the extracellular domains of transmembrane receptors with their physiological ligands are also described. Finally, the palindromic patterns of glycosylation that can occur with these expression systems, in terms of the role and location of the sequon in the primary structure, the number of mannose units and the types of oligosaccharides incorporated as Asn- or O-linkages and their impact on the thermostability and immunogenicity of the recombinant protein are considered. Procedures to prevent glycosylation through manipulation of cell culture conditions or via enzymatic an...

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“…Our in vitro digestion assays have indicated that already at pH 2.75–3.0 pepsin is no longer fully activated (76, 77). The work of Tanaka and colleagues also underlines this observation, as the activation of pepsins optimally occurs at pH 1.0–3.5 and at higher pH drops significantly (78, 79). As a consequence, peptic digestion is significantly hampered.…”

Section: Physiological Situations and Pathophysiological Events Leadimentioning

confidence: 84%

Anti‐acid medication as a risk factor for food allergy

Pali‐Schöll¹,

Jensen‐Jarolim²

2010

Allergy

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To cite this article: Pali‐Schöll I, Jensen‐Jarolim E. Anti‐acid medication as a risk factor for food allergy. Allergy 2011; 66: 469–477. Abstract An important feature for oral allergens is their digestion‐resistance during gastrointestinal transit. For some oral allergens, digestion stability is an innate feature, whereas digestion‐labile antigens may only persist in times of impairment of the digestive system. In this review, we collect evidence from mouse and human studies that besides the inherent molecular characteristics of a food protein, the stomach function is decisive for the allergenic potential. Gastric acid levels determine the activation of gastric pepsin and also the release of pancreatic enzymes. When anti‐ulcer drugs inhibit or neutralize gastric acid, they allow persistence of intact food allergens and protein‐bound oral drugs with enhanced capacity to sensitize and elicit allergic reactions via the oral route. Mouse studies further suggest that maternal food allergy arising from co‐application of a food protein with anti‐acid drugs results in a Th2‐biased immune response in the offspring. Especially, anti‐ulcer drugs containing aluminum compounds act as Th2 adjuvants. Proton pump inhibitors act on proton secretion but also on expression of the morphogen Sonic hedgehog, which has been related to the development of atrophic gastritis. On the other hand, atrophic gastritis and resulting hypoacidity have previously been correlated with enhanced sensitization risk to food allergens in elderly patients. In summary, impairment of gastric function is a documented risk factor for sensitization against oral proteins and drugs.

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Soluble expression and purification of porcine pepsinogen from Pichia pastoris

Cited by 32 publications

References 32 publications

Digestion of Nucleic Acids Starts in the Stomach

Digestion of Nucleic Acids Starts in the Stomach

Expression of heterologous proteins inPichia pastoris: a useful experimental tool in protein engineering and production

Anti‐acid medication as a risk factor for food allergy

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