Protein surface charge of trypsinogen changes its activation pattern

BMC Biotechnology

Karin Buettner¹  Thomas Kreisig¹  Norbert Sträter¹  Thole Zuchner² 

[1] Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, 04103, Germany

[2] Current address: Octapharma Biopharmaceuticals GmbH, Im Neuenheimer Feld 590, Heidelberg 69120, Germany

关键词: trypsin;    Protein engineering;    Protein-interaction;    Protein expression;    Protein design;   

Others  :  1121344
DOI  :  10.1186/s12896-014-0109-5

 received in 2014-08-20, accepted in 2014-12-11,  发布年份 2014

【 摘 要 】

Background

Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction.

Results

Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme.

Conclusion

The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.

【 授权许可】

   
2014 Buettner et al.; licensee BioMed Central.

【 图 表 】

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