High purity of human secreted phospholipase A2 group IIE in Pichia pastoris using basal salts medium comparison with YPD medium

Abstract Secreted phospholipase A2s (sPLA2s) are a group of enzymes with 6–8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.


Introduction
The mammalian secreted phospholipase A2s (sPLA2s, EC 3.1.1.4)are a group of calcium-dependent enzymes with 6-8 disulfide bonds and a conserved His-Asp catalytic dyad. [1][3] Presently, 11 members have been discovered in the mammalian sPLA2s family, including group IB, IIA, IIC, IID, IIE, IIF, V, X, III, XIIA, and XIIB. [2]he availability of sPLA2s proteins in the soluble and active form will allow their further study, such as screening the selective inhibitors, studying the receptor binding, and so on.It is well established that sPLA2 group IIA, [4] IID, [5] V, [6] and X [7] act as the "bactericidal sPLA2," playing critical roles in host defense against invading bacteria and influenza by degrading their cell membrane.Therefore, cytotoxicity might be one of the reasons for their difficult recombinant expression in the active form.
A variety of expression hosts, including bacterial, yeast, insect cells, and mammalian cells, are used for the production of recombinant proteins.Among them, Escherichia coli (E.coli) is the most prevalent organism for producing sPLA2 proteins.The full set of mouse and human sPLA2s are recombinantly expressed in E. coli but as the inclusion bodies (IBs). [8]It is an inherent problem for E. coli in the process of disulfide bond formation, ultimately leading to the mispairing of cysteines, which can cause misfolding and aggregation of proteins as IBs.Meanwhile, it is tedious, time-consuming, laborious, and expensive to recover functional sPLA2 proteins from IBs.Except for E. coli, yeast is another attractive organism for producing sPLA2 proteins, [9][10][11] due to its high cell density, strong and tightly regulated promoters, and the capability for post-translational modifications and secretion expression of heterogeneous proteins.Human sPLA2 group IB protein with the propeptide in the inactive form is expressed in the inducible expression system of Pichia pastoris. [12]roup IIE (GIIE) is known as an "inflammatory" [13] and "metabolic" [14] sPLA2.GIIE is found to be an inducible enzyme in response to proinflammatory stimuli, and enhances leukotriene production and granule exocytosis in mastocytoma cells. [13]GIIE regulates lipolysis in adipocytes, [15] and it mildly promotes adiposity and fatty liver by altering the proportion of phosphatidylethanolamine and phosphatidylserine in lipoproteins. [14]or further exploration of the functional mechanism of GIIE, we have been working on the catalytic mechanism [16- 18] and structural study [19] of human GIIE (hGIIE) over the past few years, however, the availability of the active GIIE protein is the bottleneck problem in our research.GIIE protein has been expressed as IBs in E. coli with a reasonable refolding yield at first. [20]In our work, we expressed wild type [19] and mutated [16][17][18][19] hGIIE proteins in the soluble and active form using the constitutive expression system of P. pastoris.While purifying hGIIE protein, we found that the culture medium is one key factor for the final purity of hGIIE protein.In this study, we expressed hGIIE using basal salts medium (BSM) in place of YPD medium in the shake flask, and compared the cell growth, the protein yield, and the final purity of hGIIE in these two mediums.Furthermore, protein hGIIE was successfully produced on a large-scale.This study will facilitate further research of hGIIE protein and provide references for the expression and purification of other sPLA2 members.

Plasmid construction
The gene of hGIIE without the signal peptide was synthesized by Genescript (Jiangsu, China) according to its cDNA sequence (Genbank/EMBL Accession NM_014589).Having digested with EcoRI and XbaI, the fragment was further ligated into the pGAPZaA vector.The ligated plasmid was transformed into competent E. coli Top10 (KT Life, Shenzhen, China) and selected on LB plates containing 40 lg/mL zeocin at 37 C.The positive transformants were verified by sequencing analysis (Sangon, Shanghai, China).The recombinant plasmid pGAPZaA-hGIIE was extracted using plasmid extraction kit (TIANGEN, China).

Transformation of P. pastoris with a linearized pGAPZaA-hGIIE and selection of positive expressing clone
Five micrograms of recombinant plasmid pGAPZaA-hGIIE were linearized by BlnI, and then transformed into P. pastoris X-33 (Invitrogen) by electroporation according to the manufacturer's protocol.Transformants were selected on YPDS with 100 mg/mL zeocin at 29 C for 48 h.Positive clones with the highest expression level of hGIIE protein were further selected based on the results of SDS-PAGE.

Production of hGIIE in P. pastoris
The selected clone was inoculated in 10 mL YPD media with 100 mg/mL Zeocin by shaking at 29 C, 220 rpm for 24 h as the activating seed.Then it was expanded to 100 mL YPD media with 100 mg/mL kanamycin and 100 mg/mL ampicillin by shaking at 29 C, 220 rpm for 24 h as the second seed.Production of hGIIE was conducted in a 5 L shake flask containing 1 L culture medium at 29 C, 220 rpm for 96 h.We used two kinds of culture medium to study the effect of culture medium on the production of hGIIE.One was the natural medium YPD containing 10 g/L yeast extract, 20 g/L glucose, and 20 g/L tryptone, the other one was the basal salts medium (BSM) containing 4 g/L KH 2 PO 4 (pH6.0), 4 g/L (NH4) 2 SO 4 , 0.38 g/L CaCl 2 , 18.2 g/L K 2 SO 4 , 9.4 g/L MgSO 4 Á7H 2 O, 20 g/L glucose, 20 g/L tryptone, and 1 mL/L trace element solution.The trace element solution consisted of 2.5 g/L MnSO 4 ÁH 2 O, 54.17 g/L FeSO 4 Á7H 2 O, 16.67 g/L ZnCl 2 Á2H 2 O, 0.17 g/L Na 2 MoO 4 Á2H 2 O, and 0.2 g/L biotin, which was prepared according to the reference. [21]Two milliliters of samples were fetched out every 6 h during fermentation to monitor the cell growth and protein yield.

Large-scale fermentation of hGIIE
Large-scale fermentation of hGIIE was conducted in a 10 L bioreactor (Eppendorf-NBS) by the fed-batch cultivation, with the controls for the temperature at 29 C, pH at 5.8, and aeration rate above 30% dissolved oxygen (DO).Both the initial culture medium and feeding medium were BSM mentioned above.Four hundred milliliters of seed was inoculated into the bioreactor with 5 L initial medium.After 12 h cultivation, 2 L feeding medium was fed into the bioreactor with the flow speed at 2 mL/min.pH was automatically controlled by the addition of 30% NH 4 OH, and agitation speed was controlled based on DO at a set point of 30%.The culture supernatant was collected after 61 h of fermentation.

Purification of hGIIE
The culture supernatant was centrifuged at 12,000 g for 10 min at 4 C and then filtered with 0.45 lm membrane to further remove the sediment.Using TangenX SIUS TFF Filter plate (Repligen), the culture supernatant was concentrated five times and then diluted four times with buffer (10 mM MES, pH 6.0) to reduce its ionic strength.The diluted supernatant was loaded onto a 20 mL SP FF (Cytiva) column pre-equilibrated with buffer A (20 mM MES, pH 6.0).The elution was carried out with a gradient of buffer B (1 M NaCl, 20 mM MES, pH 6.0).The protein peak was collected, and the quality of the protein was analyzed by SDS-PAGE.For the sample from shake flask fermentation, it was concentrated to a final volume of 500 mL and loaded onto a 24 mL Superdex 200 increase 10/300 gel filtration column (GE Healthcare) pre-equilibrated with buffer (20 mM Tris, 200 mM NaCl, pH8.8, 4 C) with a flow rate of 0.75 mL/min.The large-scale fermentation sample was concentrated to 5 mL and loaded onto a 120 mL Superdex 75 gel filtration column (Cytiva).The target protein was eluted at a flow rate of 1 mL/min and analyzed by SDS-PAGE.

Enzymatic activity of hGIIE
This assay was carried out using sPLA2 Assay kit (Cayman, No. 765001) on a 96-well plate at 25 C.The substrate of this assay was 1,2-Diheptanoyl Thio-phosphatidylcholine (DTPC), and it was prepared at a final concentration of 1.66 mM in the assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM CaCl 2 , 100 mM KCl, 0.3 mM Triton X-100).After hydrolysis of the thioester bond at the sn-2 position by hGIIE, free thiols are detected by 5,5-dithio-bis-nitrobenzoic acid (DTNB).DNTB was dissolved by 1 mL water to yield 10 mM DTNB in 0.4 M Tris, pH 8.0, and stored on ice in the dark.The concentration of hGIIE was altered to 0.1-0.3mg/mL to ensure the absorbance increase of 0.01-0.1 per minute.During the assay, 10 mL DTNB, 10 mL protein sample, and 5 mL assay buffer were pooled in the well.Then the reaction was initiated by adding 200 mL substrate solution to the well simultaneously.Absorbance at 414 nm was measured every 1 min for 12-15 min using a plate reader (Molecular Devices, USA).The specific activity of GIIE was calculated as follows:

Construction of hGIIE expression vector and screening of positive clones
To achieve the secretory expression of hGIIE in Pichia pichia X-33, an expression vector pGAPZaA was constructed by integrating the gene of hGIIE in frame with the a-factor secretion signal sequence (Figure 1).Then the construct linearized by BlnI was transformed into P. pastoris X-33 by electroporation.Dozens of transformants were grown on the YPD plate with 100 mg/mL zeocin after 48 h.Positive clones were inoculated into YPD medium and cultured at 29 C for 50 h.SDS-PAGE results indicated that 6/7 of the transformants had successfully expressed GIIE protein compared to the negative control (Figure 2).After cleavage of the secretory signal peptide by kex2, six amino acid residues remained in the N-terminus of the tag-free hGIIE protein.
The calculated molecular mass of hGIIE is 14.64 kDa, consistent with the result of mass spectrometry (Figure S1).Results from the peptide mass of the fingerprint further confirmed that the band on the bottom of SDS-PAGE (Figure 2) was protein hGIIE (Figure S2).
Comparison of the production of hGIIE in the shake flask using BSM with that using YPD medium Shaking flask fermentation of P. pastoris typically employs YPD medium, which is more nutritious and mixed simply with natural ingredients, compared with BSM.However, unused content in the YPD medium will probably cause trouble for the following purification step of the supernatant.In this study, we compared the cell growth and protein production of hGIIE in the shake flask using BSM with YPD medium.The fine-tuning BSM was employed in the shake flask to mimic the fermentation of hGIIE in the bioreactor.Without the fed-batch process, the extra tryptone (20 g/L) was introduced into the improved BSM in the shake flask.The initial pH of KH 2 PO 4 was adjusted at 6.5 in the shake flask to replace the real-time pH control of the bioreactor.As shown in Figure 3A, during the whole fermentation process, the cell density in the nutrient-rich YPD medium was higher than that in BSM.However, GIIE enzyme activity from BSM was higher than that from YPD in about 60 h of initial fermentation (Figure 3B).The reason might be that BSM contains many metal cofactors and a more suitable ionic buffer, which facilitates the acquisition of cysteine-rich and calcium-dependent protein of hGIIE.
With the consumption of nutrients in BSM, the enzymatic activity of hGIIE from the supernatant of BSM was increased with the same level as that from YPD at the later stage from 66 to 96 h (Figure 3B).In addition, there are distinct protein bands of hGIIE after being cultured over 36 h in both mediums (Figure 4).In conclusion, the production of hGIIE in the shake flask using BSM is a slightly higher than that using YPD medium in about 60 h of initial fermentation, and the effective duration of fermentation in BSM is about 60 h.Comparison of the purification of protein hGIIE from the shake flask fermentation using BSM with that using YPD medium The shake flask fermentation supernatant from BSM and YPD medium were purified by cation exchange chromatography (CIEX), followed by size exclusion chromatography.Before the purification process, the fermentation supernatant was concentrated five times and diluted four times with buffer (10 mM MES, pH 6.0) to remove the yellow pigment and decrease the ionic strength.Then the diluted culture supernatant was loaded onto CIEX SP FF column, and samples with UV absorption peak were collected by linear gradient elution (Figures 5A, 6A).For BSM, the purity of samples from the eluted peak 1 was improved, as shown in SDS-PAGE (Figure 5A), and its purification factor was increased to 4.12 after CIEX purification (Table 1).However, for YPD, there were two eluted peaks in the curve of CIEX (Figure 6A), no protein band but maybe the pigment appeared in peak 1, and impure hGIIE protein was eluted in peak 2 analyzed by SDS-PAGE (Figure 6A).
To further purify the hGIIE protein, samples from peak1 of CIEX for BSM and Peak2 of CIEX for YPD were respectively concentrated to 500 mL and loaded onto the Superdex 200 increase gel filtration.hGIIE from BSM was eluted at 19.3 mL (Figure 5B), suggesting that protein hGIIE existed as a monomer.The enzymatic activity of this sample was 3.45 U/mg (Figure 7A), comparable to previously reported hGIIE activity. [19,20]However, the impure sample of hGIIE from YPD was eluted with two peaks at 18.4 and 19.3 mL, respectively (Figure 6B).The band with 10 kDa on SDS-PAGE (Figure 6B) was not protein hGIIE, which was confirmed by the analysis of the peptide mass of fingerprint.The enzymatic activity of this impure hGIIE sample from Superdex 200 for YPD was 2.35 U/mg (Figure 7B).Compared with the final purified GIIE sample from BSM, the sample from YPD presented lower purity, which may be attributed to the surplus of ingredient (yeast extract) in the YPD medium (Figure S3).
The protein of hGIIE was successfully purified from the culture supernatant, using BSM instead of YPD medium in the shake flask fermentation.A dozen mutants of hGIIE [17] were obtained according to this method, which was beneficial for further understanding its functional mechanism.

Purification of hGIIE from the large-scale fermentation
Large-scale fermentation of hGIIE lasted for 61 h in the 10 L bioreactor with the fed-batch cultivation, according to the fermentation time in the shake flask.Before the purification process, 6.5 L supernatant was collected by high-speed centrifugation, then handled with the same pretreatment process as the sample from the shake flask, including filtration, concentration, and dilution.Afterward, the 5 L sample was loaded onto the SP column at the flow rate of 2.5 mL/min for 22 h.Then the peak fraction of the aim protein was eluted by linear gradient elution (Figure 8A).The sample was further purified by superdex 75, presenting the       symmetric elution peak at 92.14 mL.The purity of hGIIE was more than 90% according to the result of SDS-PAGE (Figure 8B).Sample from the large-scale fermentation was successfully used for the crystallization and structure study of hGIIE, [19] and will benefit its applied research.

Conclusion
In conclusion, we have successfully expressed tag-free hGIIE in P. pastoris.Protein hGIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography.In the shake flask fermentation, Protein of hGIIE with high purity was successfully obtained, using BSM instead of YPD medium.In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein, according to the fermentation time and purification scheme in the shake flask.This study will facilitate further research on hGIIE protein.In addition, since hGIIE is an alkaline (the isoelectric point is 8.53) and cystine-rich protein, this protocol will provide references for the expression and purification of other disulfide-rich proteins and alkaline proteins.

Figure 1 .
Figure 1.Schematic map of the recombinant expression vector pGAPZaA-hGIIE.hGIIE gene was inserted between the EcoRI and XbaI sites and fused at the N-terminus with the cleavable a-factor secretion signal (a-factor), expressed under the control of the GAP promoter.

Figure 2 .
Figure 2. SDS-PAGE analysis of hGIIE expression of picked transformants.Lanes 1-7, 10 mL of 20-times concentrated culture supernatant from different selected transformants; Lane 8, 10 mL of 20-times concentrated culture supernatant from the negative strain transformed with pGAPZaA empty plasmid; M, protein molecular weight marker (kDa).

Figure 3 .
Figure 3.Comparison of the cell growth (A) and enzymatic activity (B) of hGIIE in the YPD medium with BSM in the shaking flask.

Figure 4 .
Figure 4. SDS-PAGE analysis of samples collected at different stages of the fermentation processes in the YPD medium (A) and BSM (B).All wells were loaded with 10 mL of 20-times concentrated culture supernatant at different stages of fermentation.

Figure 5 .
Figure 5.The elution curve of the purification procedure of hGIIE from the culture supernatant of BSM in the shaking flask fermentation.(A) Cation exchange chromatography (CIEX) result of hGIIE; (B) Superdex 200 result of hGIIE.The inserts show the SDS-PAGE analysis of the peak samples.

Figure 6 .
Figure 6.The elution curve of the purification procedure of hGIIE from the culture supernatant of YPD medium in the shake flask fermentation.(A) Cation exchange chromatography (CIEX) result of hGIIE; (B) Superdex 200 result of hGIIE.The inserts show the SDS-PAGE analysis of the peak samples.

Figure 7 .
Figure 7. Absorbance times for hGIIE purified from the supernatant of BSM (A) and YPD medium (B).The specific activity was calculated according to the formula: Specific activity (U/mg) ¼ DA414 min À1 0:66 mM À1 Â 0:225 ml 0:01 ml Â sample concentration.᭡A414 min À1 is the value of the line slope.

Figure 8 .
Figure 8.The elution curve of the purification procedure of hGIIE from the culture supernatant of BSM in the large-scale fermentation.(A) Cation exchange chromatography (CIEX) result of hGIIE; (B) Superdex 75 result of hGIIE.The inserts show the SDS-PAGE analysis of the peak samples.

Table 1 .
Purification summary of hGIIE expressed in Pichia pastoris using basal salts medium and YPD medium in the shake flask fermentation.
a Total amount of samples.b Specific activity.c Purification factor.d Cation exchange chromatography.