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Heterologous expression of abaecin peptide from Apis mellifera in Pichia pastoris

Microbial Cell Factories volume 16, Article number: 76 (2017) Cite this article

Abstract

Background

Antimicrobial peptides (AMPs) are the first line of host immune defense against pathogens. Among AMPs from the honeybee Apis mellifera, abaecin is a major broad-spectrum antibacterial proline-enriched cationic peptide.

Results

For heterologous expression of abaecin in Pichia pastoris, we designed an ORF with HisTag, and the codon usage was optimized. The gene was chemically synthetized and cloned in the pUC57 vector. The new ORF was sub-cloned in the pPIC9 expression vector and transformed into P. pastoris. After selection of positive clones, the expression was induced by methanol. The supernatant was analyzed at different times to determine the optimal time for the recombinant peptide expression. As a proof-of-concept, Escherichia coli was co-incubated with the recombinant peptide to verify its antimicrobial potential.

Discussion

Briefly, the recombinant Abaecin (rAbaecin) has efficiently decreased E. coli growth (P < 0.05) through an in vitro assay, and may be considered as a novel therapeutic agent that may complement other conventional antibiotic therapies.

Background

Insects have cellular and humoral defenses in the innate immunological system. The circulating hemocytes are responsible for the first, as in phagocytosis cases and pathogens nodulation [1, 2] and the production of peptides by the fat bodies related to the second [3].

The antimicrobial peptides (AMPs) are molecules present in the immune system of multinuclear organisms, acting in the defense against invaders such as gram-positive, gram-negative bacteria and fungi [4].

The abaecin peptide, found in Apis mellifera, is one of the largest proline-rich antimicrobial peptide, with 34 amino acids containing 10 prolines (29%) and no cysteine residues. The total charge is 4+, grouped within positions 12, 13, 27, and 29. Prolines are uniformly distributed through the peptide length, preventing the α-helical conformation [5, 6].

The yeast Pichia pastoris is methylotrophic, which means a capacity of using the methanol as its only carbon source. The yeast oxidizes the methanol producing formaldehyde and hydrogen peroxide, using oxygen molecules and alcohol oxidase enzymes. The yeast produces large quantity of this enzyme, due to its low affinity to oxygen, being regulated by the alcohol oxidase 1 promoter (AOX1). The AOX1 promoter within the vector, induced by methanol, leads to expression of the gene of interest at high levels. The protein expression using the pPIC9 vector occurs in an extracellular manner, decreasing the steps between expression and obtaining the peptide of interest [7, 8].

Antimicrobial peptides were isolated from many kind of organism like vertebrates and invertebrates animals, plants, bacteria, fungi, viruses and artificially synthesized in laboratory for experiments [9]. However, our aim was to construct a synthetic gene to express the abaecin peptide from A. mellifera in P. pastoris. Our purpose was to optimize the expression of this recombinant antimicrobial peptide with biological effects, leading to a possible new drug development.

Methods

Gene design and synthesis

The recombinant peptide coding sequence was designed based on the deposited sequence in the National Center for Biotechnology Information (NCBI) NP_001011617.1, of A. mellifera’s abaecin peptide. Some nucleotides sequences were added to facilitate cloning and optimization of this protein expression in yeast P. pastoris. Two restriction sites were added: in the 5′ position for EcoRI enzyme, and another one in 3′ position for NotI enzyme. It was also added hexahistidine-tag (Histag) for expression confirmation process, and a stop codon was included as well. The peptide signal of the entire abaecin sequence was not used.

The sequence was optimized with the following parameters: length: 129, GC%: 45.08, GAATTC-TACGTTCCATTGCCTAACGTTCCACAACCTGGTAGAAGACCATTTCCTACTTTCCCAGGTCAAGGACCTTTTAACCCTAAGATTAAATGGCCTCAGGGATATCGTCGACATCACCATCACCATCACTAA-GCGGCCGC, therefore, the new abaecin sequence was deduced: YVPLPNVPQPGRRPFPTFPGQGPFNPKIKWPQGYHHHHHH. Then, to optimize the sequence for P. pastoris, the codon adaptation index (CAI) was used. It measures the extension level of differential usage of codons in highly expressed genes [10]. The measurements in this technique use the OptimumGeneTM (GenScript® Corporation) software.

After analysis and deduction of the optimized DNA sequence, the chemical synthesis of the minigene was performed, cloned in pUC57, and sequenced for confirmation (GenScript® Corporation).

Sub-cloning of the synthetic gene in the expression vector pPIC9

The insert was removed from vector pUC57 using the NotI and EcoRI enzymes. The vector pPic9 was also digested with the same enzymes, quantified and dephosphorylated with SAP enzyme. It was used 16.7 ng of the insert in the ligation reaction with 50 ng of pPIC9 vector and 1U of the DNA ligase I (Invitrogen) at 14 °C overnight. The product of this reaction was named pPic9abacin, confirmed by electrophorese at 0.8% of agarose. The Escherichia coli Top10 bacteria were transformed with the pPic9abaecin by electroporation in 0.2 cm cuvette with lysogeny broth (LB), following the parameters: 2.5 kV, 200 Ω and 25 µF, electroporated in Bio-Rad Gene Pulser (Bio-Rad). These clones were cultivated in LB medium containing 50 µg/mL of Ampicillin. The plasmid extraction was performed using QIAprep® (Qiagen®), following the kit protocol.

Transformation and electroporation of Pichia

The recombinant plasmid was linearized with SacI enzyme (FastDigest, Fermentas) 1 μL/μg (enzyme/DNA), purified with phenol/chloroform/125:24:1 and used to transform the strain GS115 P. pastoris yeast. The GS115 strain was cultivated in 50 mL of yeast extract peptone dextrose (YPD, 1% yeast extract, 2% peptone and 2% dextrose) at 30 °C overnight, then inoculated 0.5 mL of this culture in 500 mL of fresh YPD at 30 °C overnight until OD600 = 1.3–1.5. The cells were centrifuged at 1500×g for 5 min at 4 °C and resuspended, for each of the followed steps: 500 and 250 mL sterilized water, then in 20 and 1 mL of sorbitol 1 M, all processes realized in ice-cold. The transformation system of 10 µg of linearized DNA in 10 μL TE Buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and 80 µL of competent P. pastoris cells was incubated in ice-cold electroporation cuvette of 0.2 cm for 5 min in ice. The electroporation followed the parameters of 1.5 kV, 200 Ω and 25 µF, cells were immediately incubated with 1 mL of 1 M sorbitol and spread 200 μL of aliquots on RDB plates (regeneration dextrose base, 9.3:1 sorbitol/agar, w/w) without histidine. The plates were incubated at 30 °C until colonies appear [8]. The transformation was confirmed by PCR and electrophorese in agarose gel 1.5% of the clones.

Expression of the recombinant peptide

A recombinant colony was inoculated in buffered minimal glycerol (BMG) medium containing 100 mM potassium phosphate, pH 6.0, 1.34% (w/v) of yeast nitrogen base (YNB), 4 × 10−5% (w/v) of biotin, and 1% glycerol, for two days at 28 °C. Then, the cells were centrifuged and re-suspended in buffered minimal methanol (BMM) medium, composed of 100 mM of potassium phosphate, pH 6.0, 1.34% (w/v) YNB, 4 × 10−5% (w/v) biotin, and methanol 0.5%, in order to avoid the action of proteases [11]. at the optical density OD600 = 1.0, under the temperature of 28 °C, at 250 rpm.

To better verify the induction time of expression rates, supernatants were collected at times 0, 6, 12, 24, 36, 48, 60, 72 and 96 (h) with the addition of 0.5% (w/v) of methanol at times 0, 24, 48 and 72 (h). The expression confirmation was analyzed by Tricine-SDS-PAGE electrophoresis [12], and silver-stained.

Growth inhibition test

The inhibition test was done in 96-well microtiter plate containing, in the first row, 200 µL of LB, as negative control. A mix containing 200 µL of LB with E. coli DH5α, in an OD595 = 0.3, was added in the second row as first positive control (only E. coli). In the third, fourth and fifth rows was added 200 µL of LB with E. coli and new lyophilized BMM medium in quantities of 1, 10 and 25 µg, respectively, control for supernatant without recombinant peptide (named BMM). In the sixth, seventh and eighth rows were added 200 µL of LB with E. coli and BMM medium with abaecin in quantities of 1, 10 and 25 µg, respectively. The test was run in quadruplicated and the average of OD values from the negative control were subtracted from all wells.

Abaecin peptide modeling

The abaecin peptide was modeled by ab initio method, which is used when there is little or no initial information about the molecule structure in the data banks. The Rosetta 3.5 software [13], specifically the protocol of the AbinitioRelax software [14,15,16,17,18,19] was used to this goal. Approximately 2000 models were generated, one being selected after evaluation and validation using the dDFIRE [20]. The dDFIRE generates a score based on the free energy of the structure, therefore we select, among the thousands of models, one who has minor free energy. The peptide was visualized using the Chimera 1.1 software [21], where views of surface of the Coulombic electrostatic potential and amino acids hydrophobicity were generated.

Statistical analysis

The data obtained was analyzed using the Prism 4.0 software (GraphPad, San Diego, CA). After verification of data distribution, it was used analysis of variance for repeated variables, and Bonferroni tests to compare the obtained p value (P < 0.05).

Results

Gene design and synthesis and codon optimization

After the deduction and optimization of the new gene sequence, the abaecin of A. mellifera and the modified abaecin sequences were aligned by Clustal W software to check the non-modified regions and the optimized codons. The chemically synthesized gene of abaecin, with the size of 143 bp, was cloned into pUC57 clone vector.

Expression of recombinant peptide

The colonies of P. pastoris transformed with pPic9abaecin were grown in BMG medium for development and then in BMM for expression. Supernatants were collected from the expression medium in different times (Fig. 1). At times T3 (72 h) and T4 (96 h), it is possible to visualize the 5.2 kDa band showing that the selected colony produced the isolated peptide, not excluding the possibility of also containing peptides in the truncated form.

Fig. 1
figure 1

Electrophoresis Tricine-SDS-PAGE, silver-stained. Expression time: T1 (24 h), T2 (48 h), T3 (72 h), T4 (96 h) with addition of 0.5% of methanol at all times. It shows the production of the peptide abaecin, of approximately 5.2 kDa, migrating lower than the last band of the marker (M) of 6.5 kDa. The beginning of the peptide’s expression happened at the 72 h of incubation under 30 °C

Full size image

Inhibition growth test

Due to non-purification of the peptide in the present study, we adapted the antibiogram assay using the crude expression supernatant. After the confirmation of the production of recombinant peptide, the supernatant containing peptide was lyophilized and tested in E. coli culture in 96-well microtiter plate for antimicrobial activity confirmation.

The test showed that the quantities of 10 and 25 µg of lyophilized medium, containing the recombinant peptide, was sufficient to significantly inhibit the E. coli growth after 24 h treatment (Fig. 2b, c). There was no interference in the abaecin peptide inhibition by the lyophilized BMM medium. The quantity of 1 µg did not inhibit bacterial growth (Fig. 2a).

Fig. 2
figure 2

Graphics of optical density (OD) measurements by time in the analyzed wells. Square E. coli (first positive control); triangle BMM (LB with E. coli and new lyophilized BMM medium without recombinant peptide); and circle abaecin (LB with E. coli and BMM medium with recombinant abaecin). The graphics a–c show three different quantities of lyophilized BMM medium with and without abaecin, 1, 10 and 25 µg, respectively. *Significant difference compared to BMM sample (P < 0.01, Bonferroni test) and abaecin (P < 0.001, Bonferroni test) at time 24 h

Full size image

The statistical test showed significant difference in rAbaecin activity in relation to controls (E. coli and BMM only). It presented antibacterial activity in concentrations of 10 and 25 µg, respectively, after time 12, accentuating after 24 h.

Abaecin structure

The peptide sequence optimized abaecin (43 aas + histag) showed a α-helix structure in C-terminal portion composed by (KWPQGYHHHHHH) residues in red on structural analyses. The views of surface of the Coulombic electrostatic potential and amino acids hydrophobicity showed in (Fig. 3). This conformation suggests an interaction with the bacterial membrane.

Fig. 3
figure 3

Structural analysis of the heterologous peptide. In a structure abaecin with α-helix in red, in b analysis of electrostatic surface: positive charges in blue and negative charges in red, and c hydrophobicity surface analysis, warmer colors indicate hydrophobic regions

Full size image

Discussion

The antimicrobial effect of peptides, and their production, has been studied in the immunological system of animals, such as bees. A study on the bee specie Bombus pascuorum identified four types of antimicrobial peptides: defensin, hymenoptaecin, apidaecin and abaecin. These peptides were directly extracted from the animal’s hemolymph, which were purified and tested in bacterial culture, presenting antimicrobial activity [22].

In wasps from the specie Pteromalus puparum an abaecin cDNA was identified. The protein was chemically synthetized and tested in E. coli culture using this cDNA sequence, showing inhibitory activity, and no hemolytic activity was shown [23].

Previous DNA recombination methods were heavily used to high levels of peptide expression [24, 25]. For a long time, the system of proteins expression by E. coli was used due to its easy maintenance and fast culture growth [26]. It was not possible to use E. coli as an expression system because the recombining product is toxic to the bacteria. Because of that, the P. pastoris expression system was used.

The P. pastoris yeast, being a eukaryote, has advantages in the protein expression and processing, being able to make post-translational modifications. Another great advantage is the fact that, it is easier to manipulate when compared to both E. coli and Saccharomyces cerevisiae expression. The methodology used is also faster and cheaper compared to other expression systems like baculovirus or other culture tissues, allowing a high expression index [8, 27, 28].

The use of P. pastoris yeast has been used as an important protein expression method. Studies of antimicrobial peptides based on shrimp [29], butterfly, frog [30], Drosophila melanogaster, spine soldier bug [31] and human [32] genes have proven the effectiveness of this system for the production of these peptides without affecting their inhibitory activity. Here we describe a production of recombinant abaecin (from A. mellifera) in P. pastoris host.

The chemical synthesis of the abaecin gene from A. mellifera, to express the peptide in P. pastoris yeast, allowed the production of the antimicrobial peptide. This system, which has shown capacity of a fast production and easy maintenance, can be used to deliver the expected results. The use of pPIC9 vector of expression allowed the peptide to be expressed in an extracellular way, reducing the number of steps in the manipulation methodology. By using the expression optimization, it was possible to estimate the necessary time for the production.

In order to the abaecin peptide be tested in E. coli samples, the optimum incubation period was 24 h. After this time the OD of the analyzed samples decreases, supposedly because bacterial growth reaches a high concentration, causing bacterial death due to the lack of supply and low environmental conditions in the culture mediums [33]. The treated samples with the abaecin in the lyophilized BMM medium peptide at 10 µg, showed an antibacterial activity after 12 h keeping an OD lower than 0.2. On the other hand, the sample treated with 25 µg showed antibacterial activity profile between 6 and 12 h with an OD close to 0.1 (Fig. 2).

The antibacterial activity depend on the hydrophobicity and the structural ability to assume an amphipathic helical conformation [34, 35]. Other patterns affect the potency and spectrum of the AMPs α-helix, such as sequence, charge and amphipathicity. These features are interrelated and they are the key for the development of more potent AMPs with a straight action [36, 37].

Antimicrobial peptides with more than 20 aas proline rich, like abaecin, showed effectiveness against gram-positive bacteria and fungi [38,39,40]. In another study, the chemically synthetized abaecin peptide, based on B. pascuorum, didn’t show any effect without the aid of another peptide. When combined with other peptide, it exhibited action against E. coli growth [41]. Our heterologous abaecin, expressed in the P. pastoris system, showed antibacterial activity without the use of another AMP.

The charge of the proline-rich AMPs peptides (verified by in silico modeling) may provide the crossing of the membrane cell in non-lytic manner, interacting with proteins from the cytoplasm of the bacterium hampering protein synthesis [42]. This process shortens the quantity of the total bacteria in the samples, showed by the decreasing levels of the OD in the reading 595 nm in the test (Fig. 2).

It was shown that the methodology used facilitated the results of the proposed study. The chemical synthesis based in the sequence of the abaecin gene of A. mellifera, and optimized to P. pastoris (pPic9abaecin) proved to be efficient inside the expression system. The pPIC9 vector was of great importance to simplify the steps of the process, enabling it to obtain the peptide abaecin in the supernatant expression medium. The rAbaecin expressed in a heterologous manner at time 72 h, showing that there is an inhibition variation of microbial growth of E. coli DH5α bacteria within both time 6, 12 and 24 h.

Further studies are needed to the large-scale production and purification of this compound, in order to produce an antibiotic with such elements.

This results also shows that the heterologous abaecin peptide has antimicrobial activity against E. coli, and have biotechnological potential for the production of new antimicrobial drug, which acts against bacteria resistance to current drugs.

Abbreviations

AMP:

antimicrobial peptides

AOX 1:

alcohol oxidase 1 promoter

pPic9abacin:

pPIC9 vector-insert

rAbaecin:

recombinant abaecin

YPD:

yeast extract peptone dextrose

RDB:

regeneration dextrose base

BMG:

buffered minimal glycerol

YNB:

yeast nitrogen base

BMM:

buffered minimal methanol

References

  1. Jiravanichpaisal P, Lee BL, Söderhäll K. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology. 2006;211:213–36.

    Article  CAS  Google Scholar 

  2. Strand MR. The insect cellular immune response. Insect Sci. 2008;15:1–14.

    Article  CAS  Google Scholar 

  3. Arrese EL, Soulages JL. Insect fat body: energy, metabolism, and regulation. Annu Rev Entomol. 2010;55:207–25.

    Article  CAS  Google Scholar 

  4. Izadpanah A, Gallo RL, Diego S. Antimicrobial peptides. J Am Acad Dermatol. 2005;52:381–90.

    Article  Google Scholar 

  5. Chou PY, Fasman GD. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148.

    CAS  Google Scholar 

  6. Xu P, Shi M, Chen X-X. Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana. PLoS ONE. 2009;4:e4239.

    Article  Google Scholar 

  7. Romanos MA, Scorer CA, Clare JJ. Foreign gene expression in yeast: a review. Yeast. 1992;8:423–88.

    Article  CAS  Google Scholar 

  8. Pichia Expression Kit, for expression of recombinant proteins in Pichia pastoris, Catalog No. K1710-01 [Internet]. 2014. p. 98. Available from: https://tools.thermofisher.com/content/sfs/manuals/pich_man.pdf. Accessed 5 Apr 2017.

  9. Alves D, Olívia Pereira M. Mini-review: antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces. Biofouling. 2014;30:483–99.

    Article  CAS  Google Scholar 

  10. Sharp PM, Li WH. The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987;15:1281–95.

    Article  CAS  Google Scholar 

  11. Clare JJ, Romanes MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, et al. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene. 1991;105:205–12.

    Article  CAS  Google Scholar 

  12. Schagger H. Tricine-SDS-page. Nat Protoc. 2006;1:16–22.

    Article  Google Scholar 

  13. Rohl CA, Strauss CEM, Misura KMS, Baker D. Protein structure prediction using Rosetta. Methods Enzymol. 2004;383:66–93.

    Article  CAS  Google Scholar 

  14. Raman S, Vernon R, Thompson J, Tyka M, Sadreyev R, Pei J, et al. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins Struct Funct Bioinforma. 2009;77:89–99.

    Article  CAS  Google Scholar 

  15. Bradley P, Misura KMS, Baker DB. Toward high-resolution de novo structure prediction for small proteins. Science. 2005;309:1868–71.

    Article  CAS  Google Scholar 

  16. Bonneau R, Strauss CEM, Rohl CA, Chivian D, Bradley P, Malmström L, et al. De novo prediction of three-dimensional structures for major protein families. J Mol Biol. 2002;322:65–78.

    Article  CAS  Google Scholar 

  17. Bonneau R, Tsai J, Ruczinski I, Chivian D, Rohl C, Strauss CEM, et al. Rosetta in CASP4: progress in ab initio protein structure prediction. Proteins Struct Funct Genet. 2001;45:119–26.

    Article  Google Scholar 

  18. Simons KT, Ruczinski I, Kooperberg C, Fox BA, Bystroff C, Baker D. Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins. Proteins Struct Funct Genet. 1999;34:82–95.

    Article  CAS  Google Scholar 

  19. Simons KT, Kooperberg C, Huang E, Baker D. Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. J Mol Biol. 1997;268:209–25.

    Article  CAS  Google Scholar 

  20. Yang Y, Zhou Y. Specific interactions for ab initio folding of protein terminal regions with secondary structures. Proteins Struct Funct Genet. 2008;72:793–803.

    Article  CAS  Google Scholar 

  21. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–12.

    Article  CAS  Google Scholar 

  22. Rees JA, Moniatte M, Bulet P. Novel antibacterial peptides isolated from a European bumblebee, Bombus pascuorum (Hymenoptera, apoidea). Insect Biochem Mol Biol. 1997;27:413–22.

    Article  CAS  Google Scholar 

  23. Shen X, Ye G, Cheng X, Yu C, Altosaar I, Hu C. Characterization of an abaecin-like antimicrobial peptide identified from a Pteromalus puparum cDNA clone. J Invertebr Pathol. 2010;105:24–9.

    Article  CAS  Google Scholar 

  24. Reichhart JM, Meister M, Dimarcq JL, Zachary D, Hoffmann D, Ruiz C, et al. Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter. EMBO J. 1992;11:1469–77.

    CAS  Google Scholar 

  25. Lee JH, Kim JH, Hwang SW, Lee WJ, Yoon HK, Lee HS, et al. High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem Biophys Res Commun. 2000;277:575–80.

    Article  CAS  Google Scholar 

  26. Huang L, Leong SSJ, Jiang R. Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27. Appl Microbiol Biotechnol. 2009;84:301–8.

    Article  CAS  Google Scholar 

  27. Buckholz RG, Gleeson MA. Yeast systems for the commercial production of heterologous proteins. Nat Biotechnol. 1991;9:1067–72.

    Article  CAS  Google Scholar 

  28. Cregg JM, Vedvick TS, Raschke WC. Recent advances in the expression of foreign genes in Pichia pastoris. Nat Biotechnol. 1993;11:905–10.

    Article  CAS  Google Scholar 

  29. Li L, Wang J-X, Zhao X-F, Kang C-J, Liu N, Xiang J-H, et al. High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expr Purif. 2005;39:144–51.

    Article  CAS  Google Scholar 

  30. Jin F, Xu X, Wang L, Zhang W, Gu D. Expression of recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) in Pichia pastoris: purification and characterization. Protein Expr Purif. 2006;50:147–56.

    Article  CAS  Google Scholar 

  31. Sang Y-X, Deng X-J, Yang W-Y, Wang W-X, Wen S-Y, Liu W-Q, et al. Secretive expression of insect antifungal peptide-encoded genes in Pichia pastoris and activity assay of the products. Agric Sci China. 2007;6:1209–16.

    Article  CAS  Google Scholar 

  32. Chen Z, Wang D, Cong Y, Wang J, Zhu J, Yang J, et al. Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus. Appl Microbiol Biotechnol. 2011;89:281–91.

    Article  CAS  Google Scholar 

  33. Robinson TP, Aboaba OO, Kaloti A, Ocio MJ, Baranyi J, Mackey BM. The effect of inoculum size on the lag phase of Listeria monocytogenes. Int J Food Microbiol. 2001;70:163–73.

    Article  CAS  Google Scholar 

  34. Blondelle SE, Houghten RA. Hemolytic and antimicrobial activities of the twenty-four individual omission analogues of melittin. Biochemistry. 1991;30:4671–8.

    Article  CAS  Google Scholar 

  35. Blondelle SE, Simpkins LR, Pérez-Payá E, Houghten RA. Influence of tryptophan residues on melittin’s hemolytic activity. Biochim Biophys Acta (BBA)/Protein Struct Mol. 1993;1202:331–6.

    CAS  Google Scholar 

  36. Saberwal G, Nagaraj R. Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities. Biochim Biophys Acta (BBA)-Rev Biomembr. 1994;1197:109–31.

    Article  CAS  Google Scholar 

  37. Maloy WL, Kari UP. Structure-activity studies on magainins and other host-defense peptides. Biopolymers. 1995;37:105–22.

    Article  CAS  Google Scholar 

  38. Otvos L. The short proline-rich antibacterial peptide family. Cell Mol Life Sci. 2002;59(7):1138–50.

    Article  CAS  Google Scholar 

  39. Rahnamaeian M, Langen G, Imani J, Khalifa W, Altincicek B, Von Wettstein D, et al. Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens. J Exp Bot. 2009;60:4105–14.

    Article  CAS  Google Scholar 

  40. Rahnamaeian M, Vilcinskas A. Defense gene expression is potentiated in transgenic barley expressing antifungal peptide metchnikowin throughout powdery mildew challenge. J Plant Res. 2012;125:115–24.

    Article  CAS  Google Scholar 

  41. Rahnamaeian M, Cytryńska M, Zdybicka-Barabas A, Vilcinskas A. The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation. Peptides. 2016;78:17–23.

    Article  CAS  Google Scholar 

  42. Rahnamaeian M, Cytryńska M, Zdybicka-Barabas A, Dobslaff K, Wiesner J, Twyman RM, et al. Insect antimicrobial peptides show potentiating functional interactions against gram-negative bacteria. Proc R Soc Lond B. 2015;282:20150293.

    Article  Google Scholar 

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Authors’ contributions

CUV and JFA designed the experiments, DPL and JFA conducted most of the experiments. NNJ modeled the peptide structure. DPL assayed the recombinant protein expression and growth inhibition test. DPL wrote this manuscript and CUV, LRG and JFA helped to revise. All authors read and approved the final manuscript.

Acknowledgements

We would like to express our gratitude to all staff from the Laboratory of Biochemistry and Animal Toxins (LaBiTox) and Nanobiotechnology Laboratory at UFU for the support and availability of the equipment used.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Coordination of Improvement of Higher Education Personnel (CAPES); National Counsel of Technological and Scientific Development (CNPq, Grant Number 445679/2014-0) and Minas Gerais State Research Foundation (Fapemig).

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Authors and Affiliations

  1. Genetics Laboratory, Institute of Genetics and Biochemistry, Federal University of Uberlândia, 1720 Pará, Uberlândia, MG, 38400902, Brazil

    Denis Prudencio Luiz, Nilson Nicolau-Junior & Carlos Ueira-Vieira

  2. Nanobiotechnology Laboratory, Institute of Genetics and Biochemistry, Federal University of Uberlândia, 1720 Pará, Uberlândia, MG, 38400902, Brazil

    Juliana Franco Almeida & Luiz Ricardo Goulart

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Luiz, D.P., Almeida, J.F., Goulart, L.R. et al. Heterologous expression of abaecin peptide from Apis mellifera in Pichia pastoris . Microb Cell Fact 16, 76 (2017). https://doi.org/10.1186/s12934-017-0689-6

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  • DOI: https://doi.org/10.1186/s12934-017-0689-6

Keywords

Microbial Cell Factories

ISSN: 1475-2859

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