Paper Title:
Expression, Purification and Characterization of Recombinant Human Gelatin in Pichia pastoris
  Abstract

Based on the idiographic character of collagenous domain of human type III collagen, a recombinant human gelatin monomeric gene (gel) was designed and synthesized. All hydrophobic amino acids (proline excluded) were replaced by hydrophilic amino acids to improve the hydrophilic properties, and the codons encoding amino acids were optimized according to Pichia pastoris bias usage. Then a recombinant human gelatin expression vector pPIC9KG6 containing six monomeric genes ligated in the same orientation was constructed successfully. After verificated the validity of construction by DNA sequencing, the recombinant vector pPIC9KG6 was electroporated into the Pichia pastoris GS115, and Mut+ pPIC9KG6 transformants were selected on the basis of G418 resistance. Then a high-level expression strain was picked up from transformants by analyzing their recombinant protein expression levels. SDS-PAGE analysis of cell lysate and fermentation supernatant of the high-level expression strain showed that recombinant human gelatin can be expressed intracellularly and secreted expression, and its expression level reaches 16.06 g per liter. Secreted recombinant human gelatin was purified from fermentation supernatant by gel filtration chromatography. By UV spectroscopy and FTIR and SEM, it was confirmed that purified recombinant human gelatin is similar to animal-derived gelatin in protein structure.

  Info
Periodical
Advanced Materials Research (Volumes 236-238)
Edited by
Zhong Cao, Yinghe He, Lixian Sun and Xueqiang Cao
Pages
2905-2912
DOI
10.4028/www.scientific.net/AMR.236-238.2905
Citation
B. Liu, Y. T. Lei, J. Zhang, L. Hu, S. L. Yang, "Expression, Purification and Characterization of Recombinant Human Gelatin in Pichia pastoris", Advanced Materials Research, Vols. 236-238, pp. 2905-2912, 2011
Online since
May 2011
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$32.00
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