High-Level Production of a Novel Antimicrobial Peptide Perinerin in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Fusion Expression

Perinerin is a small antimicrobial peptide (AMP) isolated from an Asian marine clamworm, Perinereis aibuhitensis Grube. It shows marked activity in vitro against both Gram-negative and Gram-positive bacteria. To obtain it in large amounts, the coding sequence of perinerin was cloned into pET32a(+) vector and expression as a Trx fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lyste was separated by Ni²⁺-chelating chromatography. The purified protein was then cleaved by Factor Xa protease to release mature perinerin. Final purification was achieved by ion-exchange chromatography. Recombinant perinerin exhibited a similar antimicrobial activity to the native perinerin. These works might provide a significant foundation for the following research on the action of mechanism of marine AMPs.     

Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide

The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni²⁺-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni²⁺-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.     

Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic α-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni²⁺-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K₁₂D₃₁, Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.     

Expression and purification the antimicrobial peptide CM4 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antimicrobial peptide CM4 is a 35-residue cationic peptide. To explore a new approach for the expression and purification of CM4 in Escherichia coli, the CM4 gene was cloned into the vector pET32a to construct an expression vector pET32a-CM4. The fusion protein Trx-CM4, purified by Ni²⁺-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. Purification of recombinant CM4 was achieved by reverse HPLC chromatography, and about 1.4 mg/l active recombinant CM4 with the purity more than 98% was obtained. The recombinant CM4 showed antimicrobial activities that were similar to synthetic one. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Over-Expression in <Emphasis Type="Italic">E. coli</Emphasis> and Purification of the Human OCTN2 Transport Protein

The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni²⁺-chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni²⁺-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.     

SUMO Mediating Fusion Expression of Antimicrobial Peptide CM4 from two Joined Genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. To improve the expression level of CM4 in Escherichia coli, two tandem repeats of CM4 genes were cloned into the vector pSUMO to construct an expression vector pSUMO–2CM4. The fusion protein SUMO–2CM4, purified by Ni²⁺-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. After the cleaved sample was re-applied to a Ni-IDA column, finally, about 48 mg recombinant CM4 was obtained from 1 L bacterial culture with no less than 96% purity, which was the highest yield of CM4 reported so far.     

Production and purification of a novel antibiotic peptide,adenoregulin, from a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l^–1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni²⁺-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value.     

Production and purification of a cecropin family antibacterial peptide,hinnavin II,in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antibacterial peptide hinnavin II, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin II (hinnavin II-38-Gly, hin II) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin II was expressed in soluble form, purified successfully by Ni²⁺-chelating chromatography, and cleaved by enterokinase to release recombinant hin II (rhin II). Purification of the rhin II was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin II was obtained from 800 mL E. coli culture. The molecular mass of the rhin II determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular mass of 4,195.0 Da. The purified rhin II showed antimicrobial activities against tested E. coli K 12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of hinnavin II in its biologically active form.     

Expression of recombinant hybrid peptide hinnavin II/α-melanocyte-stimulating hormone in <Emphasis Type="Italic">Escherichia coli</Emphasis>: Purification and characterization

The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/α-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni²⁺-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.     

Serial Expression and Activity Analysis of LNK-16: A Bovine Antimicrobial Peptide Analogue

Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)₃ in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)₃ was purified by Ni²⁺-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

Expression of antimicrobial peptide LH multimers in <Emphasis Type="Italic">Escherichia coli</Emphasis> C43(DE3)

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni²⁺-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC₅₀ of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper.     

Expression and purification of antimicrobial peptide CM4 by Npro fusion technology in E. coli

Antimicrobial peptide CM4 is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant CM4 have been reported. In order to establish a bacterium-based CM4 production system, CM4 was cloned into pET28a and expressed with N^pro mutant (EDDIE) fusion. CM4 expressed as EDDIE are deposited as inclusion bodies. On in vitro refolding by switching from chemotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving CM4 protein with an authentic N terminus. Purified CM4 was separated on Ni²⁺-chelating chromatography column and cation-exchange chromatography column. Mass spectroscopic analysis indicated the protein to be 4132.56 Dalton, which equalled the theoretically expected mass. N-terminal sequencing of CM4 showed the sequence corresponded to the native protein. The recombinant CM4 exhibited the same antimicrobial and anti-tumor activity as reported previously. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant CM4 with native sequences.     

Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

A codon optimized mature human β-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l⁻¹, i.e. 0.21 g mature HBD3 l⁻¹. Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli. Revisions requested 13 December 2005; Revisions received 24 January 2006     

Anti-attacin polyclonal antibody from an in vitro derived antigen used for immunoblot to quantify attacin expressed in transgenic apple

A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l^–1) by Ni²⁺-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody.     

Expression and characterization of transforming protein E7 from cervical cancer-associated human papillomavirus type 31

E7 protein is a major oncogenic factor of human papillomaviruses (HPVs) that plays a key role in virus-associated human cervical carcinogenesis. To determine the biochemical properties of the E7 protein of high-risk HPV type 31, the gene encoding the protein was cloned into a bacterial vector, pET-32a (+), to allow expression of HPV-31E7 as a thioredoxin (Trx) fusion protein in Escherichia coli BL21 (DE3). The resulting expression level of the fusion protein reached 15 ～ 20% of the total cell protein and more than 60% of the target proteins were in soluble form upon cultivation for 6 h at 30°C in the presence of 0.5 mM IPTG. The fusion protein Trx-HPV-31E7 was effectively purified by Ni²⁺-chelating chromatography and analyzed by SDS-PAGE and Western blotting. After release from the fusion protein by enterokinase cleavage and purification to homogeneity, the recombinant HPV-31E7 (rHPV-31E7) was investigated for in vitro interaction with the pocket protein p107, which is known to interact with the amino-terminal portion of the protein. The immunoprecipitation studies revealed strong interactions of rHPV-31E7 protein with p107, suggesting it had binding activities and retained its conformational properties.     

Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27

This study reports the first successful recombinant expression of cationic antimicrobial peptides human beta-defensin-26 and human beta-defensin-27 in Escherichia coli. HBD26 and HBD27 genes were synthesized through codon optimization, and each gene was then cloned into the expression vector pET32, which feature fusion protein thioredoxin at the N-terminal. The recombinant plasmids were then transformed into E. coli BL21 (DE3) and cultured in MBL medium, which gave yields of HBD26 and HBD27 fusion proteins of up to 1.38 and 1.29 g l⁻¹, respectively. Affinity chromatography was used to purify the soluble fusion proteins, and the N-terminal TrxA tags were cleaved off by enterokinase. Pure HBD26 and HBD27 were then obtained by cationic exchange chromatography. The overall recovery of HBD26 was 38% and that of HBD27 reached 36%. Both variants showed salt-sensitive antimicrobial activity against gram-negative E. coli but not against gram-positive Staphylococcus aureus and Saccharomyces cerevisiae.     

Cloning,expression, and characterization of xylose reductase with higher activity from <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyll) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni²⁺-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 μM and 161.9 μM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat=1.44×l0⁴ min⁻¹) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L · h xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.     

Identification and characterization of a multi-domain sulfurtransferase in Phanerochaete chrysosporium

A sulfurtransferase gene (PcSft) with a coding region of 546 bp was cloned from the filamentous white-rot fungus Phanerochaere chrysosporium. The 181-amino acid protein contains a highly conserved “Rhodanese-like” domain and an ATP-binding site, with a molecular weight of 20.68 kDa. Semi-quantitative RT-PCR showed that the selective expression of PcSft was involved in secondary metabolism. The recombinant PcSFT protein was expressed in E. coli BL21 (DE3) and purified by Ni²⁺-chelating and size-exclusion chromatography. Its ATPase and sulfurtransferase (SFT) activities were indentified and characterized. PcSFT exhibited optimal SFT activity at pH 8 and 30 °C as well as stability at 20 °C and pH 8. The enzyme’s stability under different temperature and pH P. indicates a potential usefulness for the detoxification of cyanide in the environment.