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. 2015 Nov 16:15:105.
doi: 10.1186/s12896-015-0221-1.

Mouse Resistin (mRetn): cloning, expression and purification in Escherichia coli and the potential regulative effects on murine bone marrow hematopoiesis

Fangyuan Wang  1   2 , Jin Gao  3   4 , Alyssa Malisani  5   6 , Xiaowei Xi  7 , Wei Han  8 , Xiaoping Wan  9
Affiliations

Mouse Resistin (mRetn): cloning, expression and purification in Escherichia coli and the potential regulative effects on murine bone marrow hematopoiesis

Fangyuan Wang et al. BMC Biotechnol. .

Abstract

Background: Resistin (Retn) is a cytokine which has a controversial physiological role regarding its involvement with obesity and type II diabetes mellitus. Recently, murine Retn was found to be a possibly potential regulator of hematopoiesis in mice shown in the screening results of a set of gene chips which mapped the expression level of murine genes during regeneration of impaired bone marrow (BM) by 5-fluorouracil.

Results: Recombinant mice Retn was expressed in Escherichia coli and purified using ion exchange chromatography. Totally 11.4 mg rmRetn was obtained from 500 ml culture with endotoxin level less than 1.0 EU/ug. The purity of recombinant murine Resistin reached to at least 97.6% via SDS-PAGE analysis and HPLC. The protein possessed chemotaxis effects in the mouse aortic endothelial cells in vitro in transwell analysis. In vitro, rmRetn could up regulate the CFU number of mice BM and after rmRetn was administered, the cell number of murine bone marrow was significantly increased in vivo after chemotherapy. Finally, rmRetn was found able to protect mice from the chemotoxicity of 5-fluorouracil.

Conclusions: The discovery demonstrated a new function of murine Retn and suggested that it could potentially accelerate bone marrow regeneration post chemotherapy.

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Figures

Fig. 1
Fig. 1
Upregulation of Retn in BM cells after ablation. a Normal Balb/c mice were injected with 5-Fu (300 mg/kg) or PBS as control at day 0. Total BM cell numbers per leg decreased remarkably post 5-Fu injection compared to the control (P < 0.001). 6 mice (3 males and 3 females) were individually investigated at each time point in 3 independent experiments. b Gene expression analysis in the Affymetrix oligonucleotide microarrays. Total mRNA of BM cells of mice was extracted at day 0, 3, 7, 11 and 14 post 5-fluorouracil injection and mixed thoroughly for genechip hybridization. 6 mice were utilized at every time point after chemotherapy, respectively. Microarray hybridizations were performed on gene chip Affymetrix mouse genome 430 2.0. The hybridization intensities of Retn were shown at 0, 3, 7, 11, 14 days after 5-Fu treatment. The mRNA level of mResistin reached the maximum of 16.3 fold of the normal level on the 7th day after chemotherapy. c Serum samples obtained from PB were assayed for Retn by ELISA. Samples of 3 mice at each time point were analyzed individually, and great increase was detected by day 7 (*P = 0.008) and 11 (*P = 0.003) after chemotherapy, compared with the basic concentration at day 0. All values were given as mean ± SD
Fig. 2
Fig. 2
Cloning, expression, purification and characterization of rmResistin. a: Construction of rmRetn expression plasmid, and arrow indicates the PCR product, of mResistin ORF, which contains 288 base pairs and codes 94 amino acids of mature mResistin with an additional initiating codon on 5’ terminus and a stop codon on 3’ terminus. M, marker; 1, PCR product of rmResistin expression plasmid verification. b: Compared with whole bacteria proteins without induction, a protein band, as arrow indicates, appears after induction with IPTG. M, marker; 1, whole bacterial proteins prior to induction; 2, whole bacterial proteins 4 h post induction with 1 mM IPTG. c: Inclusion bodies of rmResistin were dissolved in denaturing buffer of GdnHCl (lane 1) and renatured in renaturing buffer (lane 3). The impurities and unsuccessfully renatured inclusion bodies (lane 2) were removed. M, marker; 1, solubilized inclusion bodies; 2, pellet of centrifugation post renaturation; 3, the renatured protein solution. d: Loaded on the SP sepharose Fast Flow resins, rmResistin was washed and eluted with linear NaCl concentration from 0.02-1 M and the only UV280 peak, eluted out when conductivity reached, as the arrow indicates, shows rmResistin. Absorbance at 280 nm and conductivity were detected on monitor. The elution was fractioned into fraction 1, 2 and 3. e: Three fractions were subjected to 15 % SDS-PAGE and only one band with molecular weight about 10.4 KDa was observed in the three lanes; M, molecular weight marker; 1, fraction 1; 2, fraction 2; 3, fraction 3. f: Western blotting results of rmResistin. Only one band positioned at about 10.4 KDa was obverved and this was identified as rmRsisitin monomer. No dimmers, trimers or polymers of rmResistin were found. M, Molecular weight standard marker; 1, whole bacterial proteins containing induced rmresistin by IPTG; 2, purified rmResistin
Fig. 3
Fig. 3
HPLC analysis results of purified rmRetn. a: RP-HPLC analysis of purified rmRetn was employed to detect sulphoxides, deamidates and/or impurities. Totally 28.5 μg rmRetn in 30 μl was loaded for analysis and two peaks were observed in the RP-HPLC spectrum, with the large one (1) identified as rmRetn and minor one (2) as other material. The purity by RP-HPLC was 97.6 %; b: SEC-HPLC was performed to detect possible polymers of rmRetn. Totally 47.5 μg rmRetn in 50 μl was loaded for analysis and only one rmRetn peak (1) was checked in SEC-HPLC spectrum and the purity of rmRetn was 100 %. Both spectra were detected at ultra violet (UV) wave length 280 nm and had been divided by blank control spectra respectively
Fig. 4
Fig. 4
Results of bioactivity determination and hemotopoiesis investigation of rmRetn on mouse BM. a: Migration of MAECs was accelerated by rmRetn at 100 or 1000 ng/ml after incubation of 8 h compared with control (p = 0.0049 and p < 0.001, n = 3); After incubation of 4 h, 100 ng/ml group didn’t exhibit significant difference from control group (p > 0.05, n = 3), while migrated number of 1000 ng/ml group was extremely significant compared with control group (p = 0.0034, n = 3). b: CFU numbers of BM per plate in the presence of rmRetn (100 and 1 000 ng/ml) were significantly increased compared with control (p < 0.001 and p = 0.0087, n = 3). c: Total BM cell numbers of normal mice were increased significantly after continuous administeration of rmRetn for 5 days, twice per day, at the dose of 50 and 500 μg/kg compared with control (PBS) group (p < 0.001 and p = 0.0036 respectively, n = 16) except for 5 ng/kg group (p > 0.05, n = 16). While the difference didn’t exist at day 10 after continuous administration of rmRetn at all the three doses of 7 days (p > 0.05, n = 16). d: Percentage of S phase of BM cells was raised largely after continuous administeration of rmRetn for 5 days, twice per day, at the dose of 50 and 500 μg/kg compared with control (PBS) group (p < 0.001 and p = 0.0036 respectively, n = 16) except for 5 ng/kg group (p > 0.05, n = 16). While the difference didn’t exist at day 10 after continuous administration of rmRetn at all the three doses of 7 days (p > 0.05, n = 16). e: Pretreatment and postreatment of rmRetn displayed thoroughly reverse effects on mice injected with 5-fluorouracil (300 mg/kg). Adminsteration of rmRetn post 5-fluorouracil treatment prolonged the life time of animals and saved two mice from the chemotoxicity of 5-Fluorouracil compared to the zone survival in control group (with only 5-fluorouracin injected) (p = 0.024, n = 15); However, the animals with mRetn pretreatment had a shorter life span than the control animals. Although the difference between pretreatment group and control group wasn’t statistically meaningful (p > 0.05, n = 15), the difference between pre- and post-treatment group was statistically meaningful (p < 0.001, n = 15); The result indicated that post-treatment of rmRetn could protect mice from the toxicity of 5-fluorouracil and increased their survival rate
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