Format

Send to

Choose Destination
J Agric Food Chem. 2011 Jul 13;59(13):7127-34. doi: 10.1021/jf200778r. Epub 2011 Jun 7.

New strategy for expression of recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71.

Author information

1
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China.

Abstract

Gelatin is a well-known biopolymer, and it has a long history of use mainly as a gelling agent in the food industry. This paper reports a new method for producing recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71. Three independent expression cassettes encoding for specific length of gelatin, prolyl 4-hydroxylase (P4H, EC 1.14.11.2), α-subunit (αP4H), and protein-disulfide isomerase (PDI) were individually cloned in one expression vector, pPIC9K. The modified gelatin gene and two subunit genes of P4H were under the control of two different inducible promoters, namely, alcohol oxidase 1 promoter (PAOX1) and formaldehyde dehydrogenase 1 promoter (PFLD1), respectively. The results of sodium dodecylsulfate-polyacrylamide gel electrophoresis show that a recombinant gelatin was successfully expressed in P. pastoris KM71 by methanol induction. Liquid chromatography coupled with tandem mass spectrometry analysis indicates that the expressed gelatin was hydroxylated with approximately 66.7% of proline residues in the Y positions of Gly-X-Y triplets. The results of nuclear magnetic resonance spectroscopy of recombinant gelatin test show that the (1)H and (13)C spectra have many corresponding characteristic displacement peaks, and amino acids composition analysis shows that it contains hydroxyproline and its UV absorption is consistent with the characteristics of gelatin.

PMID:
21604758
DOI:
10.1021/jf200778r
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center