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C-terminal sequence for purification. We next sought for appropriate chassis


cells for fermentation after the determination of amino acid sequence of SRHC Type XVII. Common expression systems used for recombinant protein production including prokaryotic (e.g. E. coli) expression system, eukaryotic expression system (e.g. Pichia pastoris), insect cell (baculovirus) expression system, mammalian cell expression system. Recombinant protein produced by mammalian cells involves a high expression cost and is not suitable for mass production of raw materials of cosmetics. The insect cell (baculovirus) expression system not only has a high cost and a low yield, but also shows a huge difference from human cells in post- translation. Apart from these two methods,


recombinant protein obtained by the E. coli expression system has no post-translational modification and secretory expression is hard to achieve in E. coli. Thus, cell lysis is needed for the purification of target proteins. However, the cell wall of gram-negative bacteria is known as endotoxin, which requires complicated purification processes to remove, and this will lead to great increase in the cost. Taken together, we decided to select Pichia


pastoris for production of SRHC Type XVII. Secretory expression is a proven technique in yeast expression system by adding secretory peptides to the coding sequence.10 Besides, Pichia pastoris have the capacity of post-translational modification, and this is essential for the maintenance of bioactivity of recombinant proteins originated from human proteins.


Scale production by biological fermentation with Pichia pastoris of SRHC Type XVII We first optimized the base sequence of SRHC Type XVII according to the codon usage


A B


170801 Marker kDa Marker 170801 190


140 100 70


50 42


32 25


17649.6 33312.2 Mass 48974.8 64637.4


Figure 2: Verification of successful secretory production of SRHC Type XVII in Pichia pastoris. A: SDS-PAGE result of fermentation supernatant from 170801. 170801 was the name for one of the engineered yeast cell during the experiment. B: Western-blot result of fermentation supernatant from 170801 (treated with anti-His tag antibody). C: Western-blot result of fermentation supernatant from 170801 (treated with anti-strep tag II antibody). D: MALDI-TOF MS result of purified SRHC Type XVII. E: FT-IR results of purified SRHC Type XVII. F: Growth curve of engineered Pichia pastoris and corresponding protein expression level determined by UV quantification under scale fermentation condition


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kDa 190


70 50


42 32


25 C 170801 Marker kDa


140 100


190 140


100 70


50 42


32 25


2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2


4000 D 23811.6797 3500 Figure 1: Schematic illustration of plasmid used for recombinant expression of SRHC Type XVII


bias of Pichia pastoris. After that, the coding sequence was inserted into plasmid pPIC9K and the resultant plasmid pPIC9K-C170801 was electroporated into Pichia pastoris after verification by DNA sequencing (Figure 1). We tested the expression level of SRHC


Type XVII on different strains and selected the strain with highest strain (named as 170801) for scale-up fermentation. We carried a SDS-PAGE analysis of the supernatant of 170801 after fermentation and found that there was a clear single band (Figure 2A). We further adopted Western Blot to confirm


E Amide A Amide B Amide III Amide I Amide II 3000


2500 Wave number (cm-1


2000 ) 1500 1000 500


300 250 200 150 100 50 0


that the clear band is just SRHC Type XVII with anti-his tag antibody and anti-strep tag II antibody respectively (Figure 2B and 2C). The results above indicated that SRHC Type XVII could be effectively produced by secretion. MALDI-TOF MS result further verified that the precise molecular weight of SRHC Type XVII is about 23.8-kDa (Figure 2D). Characteristic peak of collagen could also be detected with FT-IR (Figure 2E). Finally, we carried the scale-up fermentation of SRHC Type XVII in 170801 and observed increased protein concentration with bacterial


F


Protein concentration ■ Bacterial concentration ■


14 12 10 8 6 4 2 0


16 20 24 28 32 36 40 44 48


101


Absorbance


OD600


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