Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin
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Insulin controls hyperglycemia caused by diabetes, and virtually all treatments require exogenous insulin. However, the product’s extensive post-translational modifications have hindered the manufacture of recombinant insulin.
Here we report a novel production method for a monomeric B22Asp desB30 insulin analog (B22D desB30 insulin). Its precursor, DPIP, is fused to an N-terminal chitin-binding domain and intein self-cleavage tag. The fusion protein is expressed and purified from E. coli and immobilized on chitin resins. DPIP is then released using an optimized pH shift and converted to mature insulin via trypsin digest. The resulting product appears monomeric, > 90% pure and devoid of any exogenous enzyme.
Thus, biologically active insulin analog can be efficiently produced in bacteria and potentially applicable in the treatment of human diabetes.
KeywordsIntein B22D desB30 insulin Self-cleavage Optimization
B22Asp desB30 insulin
- B22D-PIP/ DPIP
Insulin precursor of B22D
Chitin binding domain
Chitin binding domain- intein1 tag
Size exclusion chromatography
Diabetes mellitus in human causes elevated blood sugar levels for a prolonged period of time . If untreated, the disease may progress into many life-threatening complications, like kidney disease, blindness, and amputations . Virtually all patients require regular intake of exogenous insulin, which lowers their blood glucose concentration. Studies suggest that the disease affects hundreds of millions of people, with an annual cost rapidly approaching one trillion dollars .
Since 1977, production of animal insulin has gradually exited the market as recombinant human insulin emerged . However, since the human insulin monomers readily aggregate into multimers, the recombinant wild type human insulin displays a delay in treating hyperglycemia . Therefore, its monomeric analogs, termed fast-acting insulins, are developed and clinically proven to match human endogenously produced insulin [4, 6]. However, both the manufacturing process and composition of these patented insulin analogs are closely guarded commercial secrets. Due to the increasing diabetic population and rapidly rising cost, the market urgently requires the development of low-cost, fast-acting insulin analogs, especially in the underdeveloped and developing nations.
Here we report recombinant production of a fast-acting insulin analog, termed B22D desB30, matured from a single-chain precursor. In vivo assays have shown that analogs of B22D desB30 stays monomeric and exhibits 30–40% activity, compared to the human endogenous counterpart [7, 8, 9]. However, structural changes that prevented multimerization also possibly destabilized its overall folding and hence made production difficult . Therefore, we opted to recombinantly express and purify the B22D desB30 precursor by protein splicing, which yielded single chain insulin precursor (B22D-PIP or DPIP).
Protein splicing involves the precise excision of an intein from a primary translation product concomitant with the ligation of the exteins via a peptide bond . This autocatalytic process occurs post-translationally, without other enzymes or any cofactors [12, 13]. Several biotechnological applications explore the splicing properties of inteins, including protein purification, peptide cyclization and protein labeling [14, 15, 16]. Among them, the intein mediated purification with an affinity chitin-binding tag (two Intein or IMPACT-TWIN by New England Biolabs) provides a low-cost, convenient system, in which the target protein is immobilized by a single-step affinity enrichment and collected by intein self-cleavage that removes the affinity tag [17, 18]. Using this process, we obtained the native protein without any affinity tag or exogenous proteases. Subsequent tryptic digestion produced pure, monomeric B22D des30 insulin analog.
Results and discussion
CBD-intein1-DPIP fusion protein expresses in the inclusion body
The recombinant fusion protein is consistently expressed at a high level (Fig. 1b, lane 2). Most likely the target protein collects in the inclusion body and is insoluble. The fusion protein expresses well in the inclusion bodies and was insoluble, consistent with precious studies [20, 21]. After collecting the pellet after cell lysis, the fraction that redissolved after urea treatment is enriched with the 36 kDa target protein (Fig. 1b, lane 4). Solubilizing the inclusion bodies with urea allows the ready purification of the fusion protein to near homogeneity. Subsequent affinity chromatography traps nearly all renatured fusion protein on the chitin matrix. All renatured protein binds to the column after dialysis, which suggests that the re-naturation process correctly restored native protein folding (Fig. 1b, lane 5). The CBD-intein tag also allows a pH-induced auto-cleavage at the N-terminus of the insulin precursor, releasing the peptide without exogenous protease or tags.
Since intein cleavage is sensitive to both temperature and pH, we aim to fine-tune the reaction condition to maximize purification efficiency. This allows the most efficient release of the target DPIP with simple temperature and pH shift. By carrying out the cleavage reaction at three different temperatures (10 °C, 25 °C and 30 °C) and eight pH conditions (4.5 to 8.0, 0.5 interval), our study shows that the intein self-cleave reaction is strictly temperature- and pH-dependent.
This observation coincides with previous results [19, 23] in which the intein C-terminal cleavage rate increased when the pH was lowered to 6.0. Therefore, we speculate that this phenomenon maybe general, and acidic conditions may benefit the self-cleavage reaction of other intein fusion proteins. Our data suggest that this particular C-terminal intein cleavage is perhaps catalyzed by a general acid (hydronium) via alternative mechanisms. However, at extremely acidic environments (pH < 5.5), we suspect that DPIP (pI = 4.9) may become unstable or insoluble. Therefore, we determine that the optimal cleavage pH for CI-DPIP is 5.5. Previous studies suggest that intein may undergo different reaction mechanisms at varying pHs. However, no previous study has investigated intein cleavage conditions below pH 6.0 either experimentally or theoretically. Theoretical approaches proposed that at lower pH (6.0), especially below the pKa of histidine (6.04), the C-terminal cleavage reaction may take a different reaction path and increase its reaction rate . Their result suggests that N-protonation of the scissile peptide bond likely starts the C-terminal cleavage reaction, in contrast to a nucleophilic side chain cyclization mechanism proposed at physiological pH . Our results support the acid-catalyzed reaction mechanism hypothesis at low pHs.
While having very little effect on cleavage efficiency at higher pHs, temperature significantly increases the reaction rate at pH 6.5 and lower. Among all three temperature conditions tested, 25 °C shows, on average, 40% higher activity than 10 °C and 30 °C at pH 6.5–4.5. Although higher temperatures usually produce higher reaction rates, CI-DPIP cleaved inefficiently at 30 °C, similar to 10 °C. This result suggests that the intein structure in this construct may be unstable and the active site perturbed at 30 °C.
Our above data suggest that a simple pH change at 25 °C can induce the release of tag-less DPIP from the recombinant protein. At pH 7.5–8.0, the cleavage rate is the slowest (below 5%), which means that the precursor can be stored safely under these conditions. Once shifted to 25 °C and pH 5.5, the intein self-cleavage reaction rate constant increased significantly to 30%. Therefore, we conclude that the optimal temperature and pH for CI-DPIP cleavage in this study is 25 °C and pH 5.5. By shifting the buffer pH from 8.0 to 5.5, we can release pure insulin analog precursor DPIP from the column.
Intein cleavage increases over two days
Urea inhibits intein self-cleavage
DPIP precursor generates pure B22D desB30 insulin analog
Summary of DPIP Purification
Refolding of fusion protein
Chitin column and intein-mediated cleavage
Buffers used in protein purification
50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl
50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl,1% Tween
50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 1%
Tween, 4 M Urea
50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 3% Tween
50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl,0.5%
50 mM Tris-HCl, 1 mM EDTA, 10 mM β-
Mercaptoethanol, 2 mM Sodium deoxycholate pH 8.0, 8
50 mM Tris-HCl, 0.5 mM EDTA,50 mM NaCl, 5%
Glycerol, 1% Glycine, 0.1% Glutathione pH 8.0
20 mM Tris–HCl, 200 mM NaCl and 1 mM EDTA,
90% HPLC grade acetonitrile in H2O, 0.08% TFA
Since misformed disulfides will induce lower solubility [25, 26, 27], faster degradation [28, 29], and unwanted aggregation or precipitation [30, 31], our final product displayed excellent solubility and superior stability, compared to commercially available insulin (Fig. 7), indicating no evidence of any misformed disulfides. This result also agrees with previous studies [32, 33] where similar recombinantly expressed proinsulin all folded correctly.
This study aims to explore a cheaper alternative for producing monomeric human insulin analog to treat diabetes. Compared to the chemical synthesis and yeast preparation, our recombinant monomeric B22Asp desB30 insulin analog is fused to an N-terminal chitin-binding domain and intein self-cleavage tag. The fusion protein binds to chitin resins and is subsequently washed off. Next, trypsin digest releases mature insulin. The resulting product appears monomeric, > 90% pure and devoid of any exogenous enzyme. Thus, biologically active insulin analog can be efficiently produced in bacteria and potentially applicable in the treatment of human diabetes.
All the buffers used in this study are summarized in Table 2.
The coding sequence of the insulin precursor (PIP) was used as a template for subsequent site-directed-mutagenesis amplification (B22D) with Pfu under standard conditions using the primer pair M1/M2 (sequence: 5′- TTGGTCTGTGGTGAAGACGGTTTCTTCTACACC-3′ and 5′-GGTGTAGAAGAAACCGTCTTCACCACAGACCAA-3′). The coding sequence of B22D desB30 was flanked by 5′ NcoI and 3′ PstI restriction sites for inserting into the pTWIN1 (New England Biolabs) expression vector with the primer pair P1/P2 (sequence: 5′-ATATCCATGGGC AAGTTCGTCAACCAACA-3′ and 5′-ATATCCATGGGCAAGTTCGTCAA CCAACA-3′). P2 inserted an additional lysine residue at the N-terminus of DPIP to introduce a trypsin cleavage site. The recombinant plasmid was subsequently transformed into E. coli DH5α cells and confirmed by colony PCR and sequencing (Invitrogen Biotechnology Co., Ltd.). Sequences of the primers for colony PCR were primer F and R (5′-ACTGGGACTCCATCGTTTCT-3′and 5′-ATATCTGCAGCTAGTTACAGTAGTTCT-3′). The sequence-confirmed recombinant plasmid was named pTWIN1-DPIP. The design of the recombinant protein and purification scheme are illustrated in Fig. 1.
All expression experiments were performed in duplicate with good reproducibility. First, pTWIN1-DPIP were transformed into E. coli strain BL21 (DE3) and grown to optical density (A600) of 0.5, in 300 ml of LB media (2% tryptone, 1% yeast extract, 2% NaCl, w/v) containing 100 μg/ml ampicillin at 37 °C. Next, isopropyl 1-thio-β-D-galactopyranoside (IPTG) was added to the final concentration of 0.1 mM to induce expression for 4 h at 37 °C. The cells were harvested by centrifugation at 3000 g for 15 min at 4 °C and stored at − 80 °C.
All subsequent steps in this section were performed on ice or at 4 °C unless otherwise mentioned. Cell pellets were resuspended in ice-cold PBS (pH 8.0) at a ratio of 1:10 (w/v) and lysed by sonication. Since the DPIP is enriched in the insoluble fraction, the cell extracts were centrifuged at 10,000 g for 30 min at 4 °C to remove the soluble fraction. The pellets were washed sequentially with buffer A-E before redissolved thoroughly in ice-cold buffer F. Next, the mixture was dialyzed against TGE buffer (pH_8.0) with different concentrations of urea (6 M, 4 M, 2 M, 1 M, and 0 M). The insoluble fraction was removed by centrifugation at 10,000 g for 30 min.
Then the supernatant was applied to pre-equilibrated chitin affinity resins (resin diameter = 50–70 μm, New England Biolabs). The on-column cleavage of the intein fusion protein was conducted in 2 bed volumes of TGE buffer with a pH gradient (8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5) at desired temperatures (10 °C, 25 °C, 30 °C). The flow through was collected and analyzed by 12% SDS-PAGE and 16.5% Tricine -SDS-PAGE.
Quantifying the intein splice efficiency
Premature cleavage was estimated by quantifying the scanned SDS polyacrylamide gels with Bandscan 5.0 (Glyko). Bands corresponding to full-length fusion protein (CBD-intein1-DPIP, CI-DPIP) and CBD-intein1 tag (CI) were quantified by scanning densitometry and normalized against their molecular weights.
Optimizing tryptic digestion
The eluate from intein splicing was digested by trypsin at 30 °C for one hour to obtain the final B22D desB30 insulin analog. We used an enzyme/substrate ratio of 1:200 (w/w). The mixture was subsequently purified by high-pressure liquid chromatography (Wasters) (C8 column, acetonitrile and trifluoroacetic acid (TFA) as mobile phases). Monomeric B22D insulin analog was eluted at 19.5-min mark with a flow rate of 1 ml/min and a buffer H gradient of 35–50% over 30 min.
To access the quality of our sample, both the commercial insulin (Insulin Injection, MW 5778) and the B22D desB30 insulin analog was subjected to size exclusion chromatography (SEC) using Superdex 75 (GE healthcare) column under standard conditions.
We thank Prof. Xiaoxia Shao from Tongji University (Shanghai, China) for determining the molecular weight by mass spectroscopy, and Dr. Lu Wang from Donghua University for proofreading the manuscript.
T. C. designed and conceived the study. B. W. and Y. P. conducted experiments and data analysis. T. C., M. Z. and C. L. drafted the manuscript. Y. Z., C. L., A.A.S. and G. W. S. critically revised the manuscript. C. L. was the senior author providing intellectual input and supervision as well as the funding for the project. All authors read and approved the final manuscript.
The design of the study, collection, analysis, interpretation of data, and in writing the manuscript was shared equally by the following funding: Shanghai Science and Technology Committee (19ZR1471100, 19410741800), Fundamental Research Funds for the Central Universities (19D210501, 19D310517), the National College Student Innovation Experiment Program (105–03-0178028, 105–03-0178029, 105–03-0178229, 105–03-0178139).
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
Consent for publication
The authors provide consent for publication.
The authors declare no Conflicts of Interest.
- 3.International Diabetes Federation. IDF Diabetes Atlas, 9th edn. Brussels, Belgium: 2019. Available at: http://www.diabetesatlas.org. Accessed 9 Oct 2019.
- 4.Moses SG, Kapur A. Animal insulins. J Assoc Phys India. 1997;(Suppl 1):41–4.Google Scholar
- 17.Pirzer T, Becher K S, Rieker M, et al. Generation of potent anti-HER1/2 immunotoxins by protein ligation using split inteins. ACS Chem Biol. 2018. [CrossRef] [PubMed].Google Scholar
- 27.Singh S, Akhter MS, Dodt J, Sharma A, Kaniyappan S, Yadegari H, et al. Disruption of structural disulfides of coagulation FXIII-B subunit. Functional Implications for a Rare Bleeding Disorder Int J Mol Sci. 2019 Apr;22:20(8).Google Scholar
- 31.Hawk LML, Pittman JM, Moore PC, Srivastava AK, Zerweck J, Williams JTB, et al. β-Amyloid model core peptides: effects of hydrophobes and disulfides. Protein Sci 2019 Nov 11.Google Scholar
- 32.Zhang H, Chen Y, Feng YM. A recombinant monomeric human insulin mutant with resistance to trypsin design, preparation and characterization. Acta Biochim Biophys Sin. 2000;32(6):635–6.Google Scholar
- 33.Mikiewicz D, Bierczyńska-Krzysik A, Sobolewska A, Stadnik D, Bogiel M, Pawłowska M, Wójtowicz-Krawiec A, et al. Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation. PLoS One. 2017 Mar 15;12(3):e0172600.CrossRefGoogle Scholar
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