Optimization Of Large Scale Fermentation Process Of Recombinant Human Elafin

Elafin which belongs to the trappin gene family is composed of 57 amino acids and predicts a molecular mass of 7 kDa. It is an acid stable peptide with an isoelectric point of 9.7.Elafin is a potent serine protease inhibitor, which has been showed to be present in human lung secretions and epithelia. Nature Elafin is released from its precursor trappin-2 by one or several unknown protease(s), and displaying antiproteinase, anti-inflammatory and anti-bacterial properties. Elafin also acts as a component of the innate immune system to protect epithelial surfaces from infection. In addition, studies on the interaction of HIV with host factors have recently highlighted a potential role of Elafin in the pathogenesis of AIDSAlthough recombinant human Elafin (rhElafin) has been successfully expressed in Pichia Pastoris (P. pastoris), it was not widely used in industry. Because large scale fermentation and purification of rhElafin was difficult, requiring several time-consuming steps that resulted in low protein yields which seriously limited the researches and clinical application of Elafin.The aim of this study is to optimize the large scale fermentation process of rhElafin.1 Transformation of P.pastoris and selection of high-level expression coloniesThe recombinant plasmid pPICZαC-elafin was linearized with Sac I and introduced into P. pastoris X33 by electroporation using a Micropulser (Bio-Rad, USA) according to the pPICZαC vector manual. Immediately after pulsing, 1 ml of cold 1 M sorbitol was added to the cuvette, and then the cuvette contents were incubated at 28℃without shaking for 1 h. Cells were plated on yeast extract peptone dextrose (YPD) agar plates for the selection of zeocin (100μg·ml-1) resistance transformants. When the transformants appeared, single clone was cultured in 10 ml BMGY medium at 28℃with shaking at 225 rpm for 24 h. The cells were collected by centrifugation and resuspended in 10 ml BMMY. Methanol was added every 24 h to a final concentration of 0.5% for 6 days. The secretion of rhElafin into the culture medium was monitored by its inhibition activity of porcine pancreas elastase (PPE). The culture medium (1 ml) was sampled per day and centrifuged at 4℃, 12500 rpm for 5 min. Cell pellet and supernatant were separated. The supernatant was used for the detection of recombinant protein and cell pellet was used for genomic DNA analysis. To get a high level of rhElafin expression, different culture parameters including pH value and induction time were optimized. The processes were the same as above except at different pH values from 3.6 to 6.4 with 0.4 intervals. The level of rhElafin expression was monitor by its inhibition activity of PPE.2 Shake flask cultureThe highest expressed recombinant strain was plated on YPD agar plate, the plates were incubated at 28℃for about 60 h. Clone was picked in regular pattern and cultured in 10 ml BMGY medium at 28℃with shaking at 225 rpm for 24 h. Then the 10 ml culture was inoculated a 5 L shake flask containing 1 L BMGY. 36 h later, when glycerol was exhausted, 5 ml methanol was added into the medium and pH value was adjusted to 5.6. After 4 days of methanol induction, the supernatant was collected and stored in 4℃for further purification by AKTA explorer 100 chromatography system.3 Expression of rhElafin in 80 L fermentorExpression of rhElafin was also examined in 30 L fermentation broth of 80 L fermentor. A stock culture of P. pastoris was grown to an OD600 of about 8 in a 5 L shake flask containing 2 L YPD. The shake flask culture was inoculated an 80 L NBS Bioflo 5000 fermenter (New Brunswick Scientific, USA) containing 30 L of fermentation basal salts medium FM21 supplemented with PTM1 trace salts (1.1 ml·L-1of stock solution) and biotin (0.4 ml·L-1 of the stock solution). The dissolved oxygen level (DO) was set at about 30%. The pH of the medium was maintained at 5.6 by automatic addition of 5 M NH4OH and 1 M phosphoric acid and 5% antifoam was injected as required. Temperature was maintained at 28℃.The fermentation growth was divided into two phases designated glycerol batch phase and methanol-fed batch phase. At the first phase, when glycerol was exhausted and a cell yield of 200 g·L-1 wet weight was achieved, as indicated by a sharp increase in the DO concentration. The second phase was initiated by starting a methanol feed containing 1.2% (v/v) of PTM1 trace salts. During the methanol induction phase, methanol was initially added at 72 ml·h-1 for 2 h and then 120 ml·h-1 for 2 h to make the culture adapt to growth on methanol, then gradually increased to 192 ml·h-1. Sampling of the culture medium was collected to analyze cell wet weight and rhElafin activity every 6 h.4 Purification of rhElafinFor large scale purification of rhElafin, 1 L supernatant of shake flask culture was purified firstly to improve the conditions of purification of rhElafin. 1 L culture broth was purified by SP Sepharose XL with equilibration buffer (20 mM sodium phosphate pH 3.0). RhElafin was eluted with a linear gradient of 0.1~1.0 M NaCl. Absorbance was monitored at 280 nm. The protein content of each peak was analyzed by its action on PPE. The eluate with activity was loaded onto SOURCETM 30 RPC by AKTA explorer 100 chromatography system. It was eluted using 50%, 75 and 100% methanol. The eluate was detected by Tricine SDS-PAGE. When the optimal conditions were obtained, supernatant of 30 L fermentation was purified at the same conditions.5 ConclusionIn this study, the engineering yeast expressing rhElafin was successfully constructed. After a series of experiments the optimal expression conditions for rhElafin large scale fermentation were obtained as follows: the optimal induction time points was about 12 h for the strain, the optimal pH was 5.6 and at 28℃, and methanol addition concentration was about 2.4~6.4 ml·h-1·L-1 broth. The culture supernatant which contained rhElafin was purified by cation ion exchange chromatography (SP Sepharose XL). RhElafin was eluted with 0.2 M NaCl solution. The result of SOURCETM 30 RPC by AKTA exploret 100 chromatography system showed that rhElafin was eluted with 75% methanol. The final expression levels were 129 mg·L-1 and we got totally 1.96 g purified rhElafin from 30 L culture medium, the purity of rhElafin reaching 95.14%.