Optimized protocol for soluble prokaryotic expression,purification and refolding of the human inhibin α subunit,a cysteine rich peptide chain

Abstract

BACKGROUND:

Inhibin A, a member of TGF- $\beta$ superfamily, consists of $\alpha$ and $\beta$ subunits. These subunits contain several cysteine residues in amino acid sequence that forms inter- and intra-subunits disulfide bonds. Due to the reducing environment of the bacterial cytoplasm, disulfide bonds formation in E.coli cytoplasm is not possible. Therefore, this can cause misfolding, aggregation and inclusion bodies formation during protein expression. As a result, the expression of inhibin subunits in E.coli produces inclusion bodies

OBJECTIVE:

We aimed at identification of an optimized protocol for expression and recovery of inhibin $\alpha$ -subunit from inclusion bodies.

METHODS:

Two vectors, four different E.coli strains, and six solubilization conditions for were used for the optimization of inhibin $\alpha$ -subunit production. Then, the solubilized proteins were purified through Ni-NTA affinity chromatography, characterized by SDS-PAGE and Western blotting (WB) using anti-his tag antibody, and refolded by dilution.

RESULTS:

The results showed that inhibin $\alpha$ -subunits were successfully expressed in both vectors and the pET22b $+$ inhibin $\alpha$ -subunit in Shuffle ${}^{\text{TM}}$ T7 strain had the highest expression; however, most of the expression was in an insoluble form. Among solubilization buffers examined, a buffer containing 2M urea with pH 12 was the best buffer to dissolve the insoluble protein. The high purity of protein was confirmed by SDS-PAGE and WB. Non-reducing SDS-PAGE demonstrating inhibin $\alpha$ -subunit refolded well.

CONCLUSION:

The current protocol is an efficient method for protocol for expression and recovery of inhibin $\alpha$ -subunit from inclusion bodies.

Keywords

Inhibin α-subunit recombinant protein expression optimization inclusion body solubilization protein refolding

1. Introduction

Inhibins are heterodimeric glycoproteins that are composed of a common 18 kDa $\alpha$ -subunit in a disulfide-linkage with one of the two analogous 14 kDa $\beta$ -subunits, namely $\beta_{A}$ and $\beta_{B}$ [1]. The two molecules are called inhibin-A ( $\alpha\beta_{A}$ ) and inhibin-B ( $\alpha\beta_{B}$ ) accordingly [2]. Inhibins and several related proteins such as TGF- $\beta$ , growth differentiation factor, mullerian inhibiting substance, bone morphogenetic protein, and activins are members of the TGF- $\beta$ protein superfamily [3].

Inhibins are principally synthesized in the sertoli and granulosa cells and mainly play a regulatory role in the synthesis and secretion of FSH and glucocorticoids [4, 5]. Inhibins participate in erythropoiesis, liver proliferation, immune function, bone formation, skin morphogenesis and cutaneous wound repair [2].

The serum level of Inhibin, used as a diagnostic marker for different physiological conditions like ovarian cancer [6, 7, 8], male and female fertility [9], Down syndrome prenatal screening, polycystic ovary syndrome, and premature ovarian failure [10, 11, 12], is measured by monoclonal antibody-based methods such as the immunosensor assay (ELISA) enzyme [13, 14]. For producing monoclonal antibody against inhibin and other experimental works, protein is needed. As inhibin is only found at very low concentrations in most accessible fluids, such as blood serum, isolation is difficult. Therefore, the production of recombinant inhibin in a simple, fast and high-efficiency system would be useful. However, due to the hydrophobic properties of inhibin, a disulfide bond in its structure and several cysteine residues, and its overexpression in prokaryotic cells formed inclusion bodies. Recovery of soluble protein from inclusion bodies need inclusion bodies isolation from the E.coli lysate, solubilization and then refolding steps. We describe here a cost-effective, simple, and efficient approach for producing soluble inhibin $\alpha$ -subunits.

2. Material and methods

2.1 Construct preparation, digestion and sequencing

The mature hINHA (residues 233–366) coding sequence (402 bp) was obtained from NCBI accession number NM_002192.2. After codon optimization, it was synthesized and inserted into BamHI/HindIII restriction sites of the pET22b vector by GENEray (China). After transformation of pET22b $+$ inhibin $\alpha$ -subunit to the E.coli DH5a competent cells, plasmid exteraction and digestion were made. Then, the inhibin $\alpha$ -subunit fragment was subcloned into the same sites of the pCold I vector. Both Plasmids were transformed to the E.coli DH5a competent cells. For constructs verification, double restriction-enzyme digestion and bi-directional DNA sequencing with universal primers of vectors were done. The pET22b and pCold1 vectors are expression vectors with C-terminal and N-terminal His-tagged, respectively.

Table 1
E. coli strains and culture conditions for expression of the human inhibin $\alpha$ subunit

Strains	Reference	Antibiotic resistance
		Ampicillin	Chloramphenicol	Kanamycin	Tetracycline
BL21 (DE3)	Novagen	100 $\mu$ g/ml	–	–	–
Origami ${}^{\text{TM}}$ (DE3)	Novagen	100 $\mu$ g/ml	–	15 $\mu$ g/ml	12.5 $\mu$ g/ml
Shuffle T7 (DE3)	Novagen	100 $\mu$ g/ml	–	–	–
Rosetta (DE3)	Novagen	100 $\mu$ g/ml	34 $\mu$ g/ml	–	–

Table 2

Composion of buffers used for inclusion body isolation and solubilization

Buffers	Composition
A	50 mM Tris-HCl, 5 mM EDTA, 1 mM PMSF, and 8 M Urea, pH8.5
B	50 mM Tris-HCl, 5 mM EDTA, 1 mM PMSF, 6 M Gnd.Hcl, pH8.5
C	50 mM Tris-HCl, 5 mM EDTA, 1 mM PMSF, and 2 M Urea, pH12
D	50 mM Tris-HCl, 0.1 mM EDTA, 0.4% Sarkosyl, 5% Glycerol, and 50 mM NaCl, pH7.9
E	5 mM Tris-HCl, 5 mM EDTA, 1 mM PMSF, 6 M N-propanol, and 2 M Urea, pH8.5
F	50 mM Tris-HCl, 5 mM EDTA, 1 mM PMSF, 6 M $\beta$ -mercaptoethanol and 2 M Urea, pH8.5

2.2 Protein expression in different E.coli strains

The constructs were transformed into four E.coli strains namely BL21 (DE3), Shuffle T7 (DE3), Rosetta (DE3), and Origami ${}^{\text{TM}}$ (DE3) for protein expression. In brief, single colonies of bacteria were grown in Terrific Broth (TB) medium (1.2% peptone, 2.4% yeast, 0.4% glycerol) with potassium phosphate buffer (potassium dihydrogen phosphate 0.231%, dipotassium phosphate 1.25%) in the presence of appropriate concentration of antibiotics (Table 1) at 37 ${}^{\circ}$ C in 200 rpm shaking incubator overnight [15]. Then, fresh TB was inoculated with 1:100 dilution of overnight cultures and was grown at 37 ${}^{\circ}$ C with vigorous shaking (200 rpm). When OD ${}_{\text{600 nm}}$ reached 0.9, the protein expression was induced with 0.5 mM isopropyl- $\beta$ -d-thiogalactopyranoside (IPTG). Induced cells harboring pColdI plasmid cells were cultured at 15 ${}^{\circ}$ C for 24 h and those harboring pET22b were cultured at 22 ${}^{\circ}$ C for 14 h. Then, they were harvested by centrifugation at 8000 g for 10 min at 4 ${}^{\circ}$ C. Cell pellets were collected, resuspended in lysis buffer I (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole and 1 mM PMSF, pH7.8) and lysed by 10 cycles of 1 min sonication (short pulses of 40 s followed by a gap of 20 s) on ice. The supernatant and pellet were separated by centrifugation at 12,000 g for 20 min at 4 ${}^{\circ}$ C. Protein expression was checked on 13% SDS-PAGE in mini-PROTEAN electrophoresis instrument (Bio-Rad Laboratories, Hercules, CA, USA) in 85 V for 1.5 h. For the visualization of protein bands, gels were stained by Coomassie Brilliant Blue R-250.

2.3 Inclusion body isolation and solubilization

To optimize the purification of the protein from Inclusion Bodies (IBs), various solubilization buffers were checked. After cell culturing and harvesting, lysis buffer II (50 mM Tris-HCl, 5 mM EDTA, 1 mM PMSF, pH8.5) was used for lysis and sonicated as mentioned above. The cell lysate was centrifuged at 20,000 g for 20 min at 4 ${}^{\circ}$ C. Then, wash buffer A (50 mM Tris-HCl, 2% Triton X-100, 5 mM EDTA and 1 mM PMSF pH8.5) was used to resuspend the pellet, followed by sonication and centrifugation for 2 times. Afterwards, the resulting pellet was washed by resuspending it in wash buffer B (50 mM Tris-HCl, pH8.5). Six buffers were used for solubilizing of the IBs at room temperature for 2 h (Table 2) with stiring [15]. Finally, 13% SDS-PAGE were used to find the best condition for IBs solubilization.

2.4 Protein purification by His-tag affinity chromatography

For protein purification, hINHA was expressed using the pET22b vector in Shuffle T7 (DE3) strain. As pre-culture, Shuffle T7 (DE3) was grown in 5 ml TB medium for 16 h at 37 ${}^{\circ}$ C. Then, the culture was diluted with 500 ml TB medium containing 100 mg/ml Ampicillin until OD ${}_{600}$ reached 0.6. Protein expression was induced by the addition of 0.5 mM IPTG. After 22 h induction at 15 ${}^{\circ}$ C, the induced cells were harvested by centrifugation at 8000 g for 10 min, resuspended in lysis buffer II and mechanically lysed by sonication on ice. The cell lysate was centrifuged at 12,000 g for 20 min at 4 ${}^{\circ}$ C; pellet containing IBs was washed and solubilized in buffer C for 2 h at room temperature. After centrifugation at 10,000 g for 15 min, the supernatant fraction was loaded onto a Ni-NTA His. Bind purification column (Novagen) and purification was done as described by the manufacturer. In brief, the column was washed with 5 volumes of wash buffer II (50 mM Tris-HCl, 2 M Urea and 1 mM PMSF, pH8.5), followed by 7 volumes of buffer II plus 20 mM imidazole, then eluted with 5 volumes of elution buffer (20 mM Tris-HCl, 2 M Ureal, and 200 mM imidazole, pH8.5). The molecular weight and purity of the fractions containing purified INHA proteins was determined by SDS-PAGE.

Figure 1.

Agarose gel analysis for double digestion of pET22b and pColdI vector using BamHI and HindIII. A. Lane 1, 1 kb marker; lane 2, double digested pET22b. B. Lane 1, 500 bp marker; lane 2, double digested pColdI.

2.5 Purity analysis by Western blot

All fractions originated from protein purification include flow-through, wash, wash plus imidazole, elution and a non-induced sample prepared in Leammli buffer and run on 13% SDS-PAGE under reducing condition. Proteins were transferred to PVDF (polyvinylidene difluoride) membrane. After blocking with 2.5% BSA in 0.05 PBS-T for overnight at 4 ${}^{\circ}$ C and washing with 0.05 PBS-T, the membrane was incubated with 0.15 $\mu$ g/ml Mouse anti-his tag antibody (Biolegend, San Diego, CA, USA) for 2 h at 25 ${}^{\circ}$ C. Then, it was washed three times with PBS-T and then incubated with HRP-conjugated goat anti-mouse IgG (H $+$ L)-HRP (1:3000) for 2 h at 25 ${}^{\circ}$ C. After three washes, detection was performed by DAB (3,3’-diaminobenzidine) according to the manufacturer’s instruction.

2.6 Protein refolding by dilution

A commonly tried but episodically successful protocol to rescue insoluble protein is to denature the protein and try to refold it in vitro. Even the most robust protocols only refold a small fraction of the input protein, and it is difficult to purify the refolded fraction. The hINHA was purified under denaturing conditions, concentrated by amicon, loaded into a 5 ml syringe. This syringe was fitted into a pump with the tip placed inside 95 mL of the refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 1.5 M urea, 10% v/v glycerol, 0.1 M arginine, 3 mM reduced glutathione, 0.3 mM oxidized glutathione, pH8.5). Following gentle stirring for 24 h at 8 ${}^{\circ}$ C, the protein pool was concentrated back to its original volume using 10 kDa MWCO amicon column (Millipore). The concentrated pool was then dialyzed against 0.2 mM sodium phosphate, pH8.5 with three buffer changes over 36 h. Confirmation of the hINHA refolding was monitored by SDS-PAGE in non-reducing and reducing condition. For preparation each sample, the refolded hINHA protein was diluted in reducing (with 2-ME) and non-reducing (without 2-ME) SDS-PAGE sample buffer.

3. Results

3.1 Construct preparation, digestion and sequencing

After digestion of pET22b $+$ inhibin $\alpha$ -subunit, the inhibin $\alpha$ -subunit fragment was subcloned into pColdI. The validation of subcloning is demonstrated by double digestion. Digestion of both constructs were done using BamHI and HindIII. These REs were expected to produce a 402 bp band of the INHA gene, 5.078 kb and 3.992 kb bands for the remaining backbone of pET22b and pColdI vectors, respectively. The RE analysis of clones gave the expected size of the two bands (Fig. 1). DNA sequencing confrmed that the INHA gene was successfully cloned into vectors in the correct orientation and there was no alteration or base substitution in the sequence of the positive clone used for the protein overexpression (data not shown).

Figure 2.

A. SDS-PAGE analysis of the pET22b-hINHA Expression in different E.coli strains. Lanes 1 and 2: insoluble (IS) and soluble (S) fraction of INHA expression in Rosetta (DE3); Lanes 3 and 4: IS and S fraction of INHA expression in Origami ${}^{\text{TM}}$ (DE3); Lanes 5 and 6: IS and S fraction of INHA expression in BL21(DE3); Lane 7: protein marker; Lanes 8 and 9: IS and S fraction of INHA expression in Shuffle T7(DE3); Calculated molecular weights for hINHA: 19 kDa. B. SDS-PAGE analysis of the pCold1-INHA Expression in different E.coli strains. Lanes 1 and 2: S and IS fraction of INHA expression in Shuffle T7 (DE3); Lanes 3 and 4: S and IS fraction of INHA expression in BL21 (DE3); Lane5: protein marker; Lanes 6 and 7: S and IS fraction of INHA expression in Origami ${}^{\text{TM}}$ (DE3); Lanes 8 and 9: S and IS fraction of INHA expression in Rosetta (DE3); Lane 10: uninduced bacteria; Calculated molecular weights for INHA: 19 kDa.

3.2 Assessment of protein expression in different E.coli strains

The encoding sequence of mature hINHA was cloned into pET22b and pColdI vectors. The constructs were transformed into several E.coli strains, including BL21 (DE3), Shuffle T7 (DE3), Rosetta (DE3), and Origami ${}^{\text{TM}}$ (DE3) and were induced for protein expression. Then, the protein expression was evaluated by the SDS-PAGE. The results indicated that hINHA protein was 19.7 kDa and was expressed almost entirely in the insoluble fraction, in the form of IBs in all tested strains. The optimum expression level of rhINHA in pET22b vector was obtained in the presence of 0.5 mM IPTG (OD 600 nm: 0.6) after 14 h induction at 22 ${}^{\circ}$ C in Shuffle T7 (DE3) strain (Fig. 2A). Also, the induction of pColdI $+$ $\alpha$ -inhibin subunit with 0.5 mM IPTG (OD 600 nm: 0.6) had the highest protein expression in Origami (DE3) strain after 24 h induction at 15 ${}^{\circ}$ C (Fig. 2B).

3.3 Inclusion body isolation and solubilization

Overexpression of hINHA in E.coli led to the formation of protein aggregation commonly called as inclusion bodies (Fig. 2). In order to obtain biologically active and soluble protein in high yield, inclusion bodies must then be solubilized and refolded in vitro. For isolation and purification of inclusion bodies, after cell lysis and centrifugation, the pellet was washed 2 times with buffer containing detergents 2% Triton X-100 to remove contaminants (Fig. 3). The washed inclusion bodies were suspended and incubated in various buffers to find the best condition. The results indicated that the highest level of soluble protein was present in buffer C containing Urea 2 M at alkali pH (Fig. 4).

Figure 3.

SDS-PAGE analysis of hINHA inclusion bodies purification from Shuffle T7(DE3). Lane1, uninduced cells; lane 2, induced cells; lane 3, inclusion bodies after cell lysis; lanes 4 to 6, inclusion body after washing steps.

Figure 4.

SDS-PAGE (A, B & C) and Western blot (D) analysis of solubilized IBs in different buffers. A. Soluble (S) and insoluble (I) fraction of buffer A; S and I fraction of buffer B; B. S and I fraction of buffer C; S and I of buffer D; C. S and I fraction of buffer E; S and I fraction of buffer F; D. Soluble fractions of buffer A, B, C, D, E, and F. M, protein molecular weight marker.

Figure 5.

Purification of hINHA. A. SDS-PAGE analysis of Purification fractions obtained from Ni-NTA chromatography. Lane 1, uninduced cells; lane 2, induced cells; lane 3, purified IBs; lane 4, IBs solubilized supernatant of buffer c; lane 5, flow through; lane 6, wash; lane 7, wash plus imidazol 10 mM; lane M, protein molecular weight markers; lanes 7–10, elution fractions. B. Western blot analysis of the hINHA protein contained in various fractions collected from Ni-NTA affinity column. Lane 1, uninduced cells; lane 2, induced cells; lane 3, purified IBs, lane 4, IBs solubilized supernatant, laneM, protein molecular weight markers; lanes 5 and 6, washes; lane 7, elution.

3.4 Protein purification and purity analysis by Western blot

After suspension of inclusion bodies in buffer C, centrifugation was done and the solubilized supernatant was loaded on Ni-NTA Column. The hINHA was eluted in column by elution buffer. The fractions were collected and run on SDS-PAGE. The elution fractions analysis on SDS-PAGE showed suitable purity of the purification step (Fig. 5A). Moreover, the results demonstrated an intense band corresponding to the estimated molecular weight of hINHA, as detected by Western blot using commercial anti-His-tag antibody (Fig. 5B).

Figure 6.

Non-reducing and reducing SDS-PAGE analysis of refolded hINHA protein. Lane 1, uninduced cells; lane M, protein molecular weight markers; lanes 2 and 3, refolded hINHA in non-reducing condition; lane 4, refolded hINHA in reducing condition.

3.5 Protein refolding

Elution buffer containing protein was concentrated by the Amicon Ultra 10 kDa molecular weight cut-off centrifugal tubes (Millipore, Bedford, MA, USA) and refolded by adding to refolding buffer via syringe pumps with 1 ml/min rate. The protein refolding was confirmed with non-reduced (without 2-ME, disulfide-reduced) and reduced (with 2-ME, disulfide-bonded) SDS–PAGE (Fig. 6). In both conditions, only one band was seen in about 19 kDa but in non-reduced SDS-PAGE condition, refolded hINHA appeared upper that implied the proper refolding.

4. Discussion

Due to its diagnostic application and other research targets, a large-scale production of recombinant inhibin A in a low cost, high speed, and high yield expression system like E.coli is needed. The purpose of this study was to establish and optimize the expression, purification, and refolding procedure for hINHA using different E.coli strains. In mature inhibin A, alpha subunit has 134 residues containing 10 cysteine residues with 3 intrachain disulfide bonds, and beta subunit has 116 residues containing 9 cysteine residues with 4 intrachain disulfide bonds [3, 16]. Expression of a cysteine-rich protein like inhibin A in the prokaryotic system is challenging since reducing environment of cytoplasm does not let disulfide bond formation [17, 18].

To solve this problem, we applied two strategies. First, we produed the protein in the periplasmic space of bacteria. Problems for heterologous protein expression in the periplasmic space are incomplete translocation across the inner membrane, proteolytic degradation, and insufficient capacity of the export machinery [19]. In addition, protein expression in periplasm space needs N-terminal signal sequence. The production of proteins that carry a signal sequence is, for yet poorly understood reasons, often toxic to the cell [20]. In the current study, we tried to express pET22b-INHA encoding pelB signal sequence in the periplasm of BL21 (DE3) strains. The results indicated that the yield of expression in periplasmic space was low (data are not shown) and the cytosolic expression fraction, as we expected, was insoluble.

Another strategy for expression of a cysteine-rich protein uses host strains with oxidative cytoplasm that provide conditions with the proper formation of disulfide bonds [15, 21]. Therefore, we tried to express hINHA in two different E.coli strains including Origami (DE3) and Shuffle T7 (DE3). These strains have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhance disulfide bond formation in the cytoplasm [15]. Shuffle host strains have a copy of the disulfide bond isomerase DsbC gene which helps in proper formation of disulfide band [22]. In addition, hINHA expression was checked in the Rosetta strain. The most important feature of Rosetta strains is the presence of specific tRNAs for rare codons in E.coli to enhance the expression of eukaryotic proteins [23]. The results showed that a high protein expression level of hINHA occurred in the Shuffle T7 (DE3) strain but most of the protein expressed as inclusion bodies.

Over-expression of a heterologous protein in hosts like E.coli leads to the formation of insoluble aggregates. Indeed, inclusion bodies are the pure expressed protein with native-like secondary structure, interacted by ionic and hydrophobic forces, without biological activity [24]. Forming inclusion bodies is a common challenge during recombinant protein production where it occurs in 70% of cases [25]. Several parameters including induction temperature, promoter strength of vectors, inducer concentration, culture media pH, non-oxidative state of cytoplasm, lack of chaperones and post-translational modifications in prokaryotic expression system had the impact on the formation of IBs [15, 26, 27, 28]. The culture temperature after the IPTG induction was set at low temperature by using pCold vectors. Also, the pH of culture media was regulated by potassium phosphate buffer in TB media.

For protein recovery from the IBs, after IBs isolation, they were dissolved by denaturing solubilization processes using solution containing high concentration of chaotropes (such as 8 M urea and 6 M guanidine hydrochloride routinely used for IBs solubilization) and mild solubilization processes using 2 M urea in the presence of several reagents such as organic solvent, detergents, reducing agents, and alkaline pH [15, 27, 29, 30].

The mechanism of action of high concentrated Urea and Gnd.Hcl for IBs solubilization breaks hydrogen bonds and denaturing native-like secondary structure of inclusion bodies completely [31]. Mild solubilization buffers containing different reagent destabilize hydrophob interaction, reduces disulfide bonds, and disruptes ionic interactions. Therefore, native-like secondary structure of IBs approximately remains intact, leading to reduce protein aggregation during refolding step and increases recovery yield of bioactive protein [15, 32]. In the current study, we applied both methods and found that 2 M urea at high pH is suitable for higher solubilization of INHA inclusion bodies.

Solubilized INHA was filtered and passed through a Ni-NTA column for purification. For refolding, we checked on-column and dilution refolding. We observed that on-column refolding is not applicable method but refolding by dilution is a proper strategy. In the refolding phase, we used L-arginine as an aggregation suppressor to enhance folding yield. Arginine prevents aggregation of partially folded intermediates [33]. Glycerol as a protein stabilizer, enhances hydrophobic interactions between protein and solvent, so it increases folding yied. To help the protein refolding, disulfide bonds were first reduced by adding DTT to the solubilization buffer. The mature hINHA, having 10 cysteine residues and three intramolecular disulfide bonds, has a high potential for the formation of non-native, inter- and intra-chain disulfide bonds during inclusion body formation. We included reduced and oxidized glutathione in the refolding buffer for generation of the correct disulfide bond. Non-reducing SDS-PAGE showed that refolding by this dilution buffer is a proper method to produce folded hINHA.

In conclusion, in the current study, we introduced an efficient method for solubilizing and refolding hINHA.

Footnotes

Acknowledgments

This work was supported by grant number 93-04-87-27089 from Tehran University of Medical Science.

Conflict of interest

None.

References

Pangas

and Woodruff

, Production and purification of recombinant human inhibin and activin, Journal of Endocrinology 172 (2002), 199–210.

Gray

P.C.

Bilezikjian

L.M.

Harrison

C.A.

Wiater

and Vale

, Activins and inhibins: Physiological roles, signaling mechanisms and regulation, in: Hormones and the Brain, Springer, 2005, 1–28.

Weiss

and Attisano

, The TGFbeta superfamily signaling pathway, Wiley Interdisciplinary Reviews: Developmental Biology 2 (2013), 47–63.

Vanttinen

Liu

Kuulasmaa

Kivinen

and Voutilainen

, Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis, Journal of Endocrinology 178 (2003), 479–489.

Meunier

Rivier

Evans

R.M.

and Vale

, Gonadal and extragonadal expression of inhibin alpha, beta A, and beta B subunits in various tissues predicts diverse functions, Proceedings of the National Academy of Sciences 85 (1988), 247–251.

Robertson

Burger

and Fuller

, Inhibin/activin and ovarian cancer, Endocrine-Related Cancer 11 (2004), 35–49.

Welt

C.K.

Lambert-Messerlian

Zheng

Crowley

W.F.

, Jr and Schneyer

A.L.

, Presence of activin, inhibin, and follistatin in epithelial ovarian carcinoma, The Journal of Clinical Endocrinology & Metabolism 82 (1997), 3720–3727.

Ala-Fossi

S.-L.

Maenpaa

Blauer

Tuohimaa

and Punnonen

, Inhibin A, B and pro-alphaC in serum and peritoneal fluid in postmenopausal patients with ovarian tumors, European Journal of Endocrinology 142 (2000), 334–339.

Luisi

Florio

Reis

F.M.

and Petraglia

, Inhibins in female and male reproductive physiology: role in gametogenesis, conception, implantation and early pregnancy, Human Reproduction Update 11 (2005), 123–135.

10.

Makanji

Zhu

Mishra

Holmquist

Wong

W.P.

Schwartz

N.B.

Mayo

K.E.

and Woodruff

T.K.

, Inhibin at 90: from discovery to clinical application, a historical review, Endocrine Reviews 35 (2014), 747–794.

11.

Munz

Hammadeh

M.E.

Seufert

Schaffrath

Schmidt

and Pollow

, Serum inhibin A, inhibin B, pro-αC, and activin A levels in women with idiopathic premature ovarian failure, Fertility and Sterility 82 (2004), 760–762.

12.

Tsigkou

Luisi

De Leo

Patton

Gambineri

Reis

F.M.

Pasquali

and Petraglia

, High serum concentration of total inhibin in polycystic ovary syndrome, Fertility and Sterility 90 (2008), 1859–1863.

13.

Groome

N.P.

Illingworth

P.J.

O’Brien

Priddle

Weaver

and McNeilly

, Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay, The Journal of Clinical Endocrinology & Metabolism 80 (1995), 2926–2932.

14.

Knight

Muttukrishna

and Groome

, Development and application of a two-site enzyme immunoassay for the determination of’total’activin-A concentrations in serum and follicular fluid, Journal of Endocrinology 148 (1996), 267–279.

15.

Nazari

Zarnani

A.-h.

Ghods

Emamzadeh

Najafzadeh

Minai-Tehrani

Mahmoudian

Yousefi

Vafaei

and Massahi

, Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1 (PLAC1), Protein Expression and Purification 133 (2017), 139–151.

16.

Antenos

Stemler

Boime

and Woodruff

T.K.

, N-linked oligosaccharides direct the differential assembly and secretion of inhibin α-and βA-subunit dimers, Molecular Endocrinology 21 (2007), 1670–1684.

17.

and Berkmen

, Production of disulfide-bonded proteins in escherichia coli, Current Protocols in Molecular Biology 108 (2014), 16.1B.1–16.1B.21.

18.

Sanglas

Bronsoms

Arolas

J.L.

Lorenzo

and Aviles

F.X.

, Recombinant expression of disulfide-rich proteins: carboxypeptidase inhibitors as model proteins, Microbial Cell Factories 5 (2006), P47.

19.

Mergulhao

Summers

D.K.

and Monteiro

G.A.

, Recombinant protein secretion in Escherichia coli, Biotechnology Advances 23 (2005), 177–202.

20.

Baneyx

and Mujacic

, Recombinant protein folding and misfolding in Escherichia coli, Nature Biotechnology 22 (2004), 1399.

21.

Nasrabadi

Rezaeiani

Sayadmanesh

Baghaban Eslaminejad

and Shabani

, Inclusion body expression and refolding of recombinant bone morphogenetic protein-2, Avicenna Journal of Medical Biotechnology 10 (2018), 202–207.

22.

Lobstein

Emrich

C.A.

Jeans

Faulkner

Riggs

and Berkmen

, SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm, Microbial Cell Factories 11 (2012), 753.

23.

Jia

and Jeon

C.O.

, High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives, Open Biology 6 (2016), 160196.

24.

Singh

S.M.

and Panda

A.K.

, Solubilization and refolding of bacterial inclusion body proteins, Journal of Bioscience and Bioengineering 99 (2005), 303–310.

25.

Yang

Zhang

Feng

Lan

Wang

and Cao

, Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method, PloS One 6 (2011), e22981.

26.

Strandberg

and Enfors

S.-O.

, Factors influencing inclusion body formation in the production of a fused protein in Escherichia coli, Applied and Environmental Microbiology 57 (1991), 1669–1674.

27.

Singh

Upadhyay

A.K.

Singh

S.M.

and Panda

A.K.

, Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process, Microbial Cell Factories 14 (2015), 41.

28.

Castellanos-Mendoza

Castro-Acosta

R.M.

Olvera

Zavala

Mendoza-Vera

García-Hernández

Alagón

Trujillo-Roldán

M.A.

and Valdez-Cruz

N.A.

, Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli, Microbial Cell Factories 13 (2014), 137.

29.

Singh

S.M.

Sharma

Upadhyay

A.K.

Singh

Garg

L.C.

and Panda

A.K.

, Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form, Protein Expression and Purification 81 (2012), 75–82.

30.

Burgess

R.R.

, [12] Purification of overproduced Escherichia coli RNA polymerase

\sigma

factors by solubilizing inclusion bodies and refolding from Sarkosyl, in: Methods in Enzymology, Elsevier, 1996, 145–149.

31.

Lee

S.H.

Carpenter

J.F.

Chang

B.S.

Randolph

T.W.

and Kim

Y.S.

, Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure, Protein Science 15 (2006), 304–313.

32.

Upadhyay

Singh

Jha

Singh

and Panda

A.K.

, Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent, Microbial Cell Factories 15 (2016), 100.

33.

Tsumoto

Umetsu

Kumagai

Ejima

Philo

J.S.

and Arakawa

, Role of arginine in protein refolding, solubilization, and purification, Biotechnology Progress 20 (2004), 1301–1308.

Optimized protocol for soluble prokaryotic expression,purification and refolding of the human inhibin α subunit,a cysteine rich peptide chain

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Material and methods

2.1 Construct preparation, digestion and sequencing

Table 1 E. coli strains and culture conditions for expression of the human inhibin α subunit

2.3 Inclusion body isolation and solubilization

2.4 Protein purification by His-tag affinity chromatography

2.6 Protein refolding by dilution

3. Results

3.1 Construct preparation, digestion and sequencing

3.3 Inclusion body isolation and solubilization

4. Discussion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
E. coli strains and culture conditions for expression of the human inhibin $\alpha$ subunit