Recombinant cold-adapted trypsin I from Atlantic cod—expression, purification, and identification

Jónsdóttir, Guðrún R.; Bjarnason, Jón B.; Guðmundsdóttir, Ágústa

doi:10.1016/j.pep.2003.09.012

Cited by 33 publications

(35 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several bands below 20 kDa were detectedin the Cryotin F fraction indicating autolysis of some of the cod proteases, as has been reported by other authors (Jónsdóttir, Bjarnason, & Gudmundsdóttir, 2004). An 8 kDa protein band observed in the Cryotin F was also observed in the FPHs indicating that some of the hydrolysates might originate from the enzymes contained in the Cryotin F solution.…”

Section: Sds-pagesupporting

confidence: 86%

Properties of hydrolysed saithe protein isolates prepared via pH shift process with and without dewatering

Halldorsdottir¹,

Hamaguchi²,

Sveinsdóttir³

et al. 2011

LWT - Food Science and Technology

View full text Add to dashboard Cite

Section: Sds-pagesupporting

confidence: 86%

Properties of hydrolysed saithe protein isolates prepared via pH shift process with and without dewatering

Halldorsdottir¹,

Hamaguchi²,

Sveinsdóttir³

et al. 2011

LWT - Food Science and Technology

View full text Add to dashboard Cite

“…Of the different cod trypsin isoenzymes trypsin I is most abundant and is the only thoroughly characterized cod trypsin whereas trypsin X has not yet been well characterized [3,4]. For unknown reasons, cod trypsin I has proven to be difficult to produce recombinantly in bacteria [7] and yeast (unpublished results).…”

Section: Introductionmentioning

confidence: 98%

Elucidation of different cold-adapted Atlantic cod ( Gadus morhua ) trypsin X isoenzymes

Stefansson¹,

Sandholt

Guðmundsdóttir

2017

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

View full text Add to dashboard Cite

Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24 kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist.One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (k cat /K m ) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.

show abstract

“…Inactive trypsinogen was quantified by subtracting the value for the activity of free proteases from the value obtained after sample activation. This spectrophotometric method using the chromogenic substrate N-␣-benzoyl-L-arginine-p-nitroanilide (BApNA) was utilized for routine process monitoring (36). One unit of trypsin is defined as the amount of enzyme that hydrolyzes 1 mol of BApNA to p-nitroaniline per minute at 25°C and pH 8.2.…”

mentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Appl Environ Microbiol

View full text Add to dashboard Cite

Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

show abstract

Recombinant cold-adapted trypsin I from Atlantic cod—expression, purification, and identification

Cited by 33 publications

References 27 publications

Properties of hydrolysed saithe protein isolates prepared via pH shift process with and without dewatering

Properties of hydrolysed saithe protein isolates prepared via pH shift process with and without dewatering

Elucidation of different cold-adapted Atlantic cod ( Gadus morhua ) trypsin X isoenzymes

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Contact Info

Product

Resources

About