High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli

The hybrid antibacterial peptide CA-MA [cecropinA(1-8)-magainin2(1-12)] with potent antimicrobial properties but no hemolytic activity is a potential alternative antibiotic. To explore a new approach for high-level expression of the hybrid peptide CA-MA in Escherichia coli, the sequence of ubiquitin (UBI) from housefly was inserted into the plasmid pQE30 to construct the vector pQEUBI. The cDNA fragment encoding CA-MA with preferred codons of E. coli was obtained by recursive PCR (rPCR) and cloned into the vector pQEUBI to express the fusion protein (His)(6)-UBI-CA-MA. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 36% of the total proteins). With (His)(6)-tag, the fusion protein was easily purified by Ni-NTA chromatography and 36 mg of fusion protein was purified from 1L of culture medium. The fusion protein was efficiently cleaved by ubiquitin C-terminal hydrolase (UCH), yielding recombinant CA-MA with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant CA-MA was purified to homogeneity by reversed-phase HPLC and 6.8mg of pure active CA-MA was obtained from 1L culture medium. Analysis of recombinant CA-MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA-MA was 2559Da, which perfectly matches the mass (2559Da) calculated from the amino acid sequence. Analysis of CA-MA by circular dichroism (CD) revealed that the secondary structures of CA-MA in water solution were 17.4% alpha-helix and 82.6% random coil but no beta-sheet. Our results demonstrated that functional CA-MA can be produced in sufficient quantities using the ubiquitin fusion technique. This is the first report on the heterologous expression of a hybrid antibacterial peptide fused to ubiquitin in E. coli.     

杂合抗菌肽CecA-Mag的人工合成及其在Pichia pastoris中的分泌表达

根据抗菌肽天蚕素A(cecropinA,CA)N端第1~7个氨基酸残基,马盖宁(magainin,M)N端第2~12个氨基酸残基,以毕赤酵母偏爱的密码子设计合成了杂合肽CA(1~7)-M(2~12)基因,同载体pPICZα-A连接后转化Pichia pastoris受体菌SMD1168,在醇氧化酶(AOX)启动子调控下,分子量约1.9kDa的CecA-Mag杂合抗菌肽获得表达,抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G-菌及G 菌均有较好的抑菌活性。初步抑菌活性测定,显示该杂合肽对金黄色葡萄球菌、耐氨苄青霉素的大肠杆菌及枯草芽孢杆菌有良好的抑杀活性。酸稳定实验显示pH为3.2时仍具有相当高的活性。热稳定性实验显示该杂合肽100℃加热5min后仍具有抑菌活性。这些特点使得重组抗菌肽CecA-mag在疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

重组毕赤酵母产青蛤Mytimacin抗菌肽的表达研究

Mytimacin是主要在无脊椎动物中表达的Macin抗菌肽家族中的一员,具有较强的抗病原微生物活性,是利用重组DNA技术开发天然抗菌剂的良好候选者。通过RT-PCR从青蛤(Cyclina sinensis)闭壳肌中克隆编码Mytimacin成熟肽的基因,经3次PCR在该基因的5’端添加Xho I限制性酶切位点和信号肽酶识别位点、3’端添加Xba I限制性酶切位点和6×His,获得目的基因"CsMm";以pPICZαA为表达载体、毕赤酵母(Pichia pastoris)X-33为工程菌,构建重组毕赤酵母X-33/pPICZαA-CsMm。通过高浓度博来霉素筛选高拷贝酵母转化子,在28℃、250 r/min条件下,使用1.5%的甲醇诱导表达72 h;使用固化金属离子亲和层析(IMAC)对表达产物进行纯化,并通过MALDI-TOF-TOF质谱分析对纯化产物进行鉴定。另外,通过涂布法和浊度法考察重组CsMm的抑菌活性。结果表明:基于X-33/pPICZαA-CsMm重组毕赤酵母的外源表达获得了表达量为25.6 mg/L的重组蛋白,经MALDI-TOF-TOF质谱鉴定其为分子量约7.8 kD的预期重组CsMm。抑菌试验证明重组CsMm对金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、大肠杆菌(Escherichia coli)和副溶血性弧菌(Vibrio Parahemolyticus)具有明显的抑菌活性。构建的重组毕赤酵母X-33/pPICZαA-CsMm能有效合成具有生物学活性的重组青蛤Mytimacin,旨为贝类来源天然小分子抗菌剂的开发提供可资参考的技术途径。     

Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8)-magainin 2(1-12) and its analogues, on their antibiotic activities and structures

A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CA-MA), incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA), has potent antimicrobial activity without toxicity against human erythrocytes. To investigate the effects of the Gly-Ile-Gly hinge sequence of CA-MA on the antibacterial and antitumor activities, two analogues in which the Gly-Ile-Gly sequence of CA-MA is either deleted (P1) or substituted with Pro (P2) were synthesized. The role of the tryptophan residue at position 2 of CA-MA on its antibiotic activity was also investigated using two analogues, in which the Trp2 residue of CA-MA is replaced with either Ala (P3) or Leu (P4). The tertiary structures of CA-MA, P2, and P4 in DPC micelles, as determined by NMR spectroscopy, have a short amphiphilic helix in the N-terminus and about three turns of alpha-helix in the C-terminus, with the flexible hinge region between them. The P1 analogue has an alpha-helix from Leu4 to Ala14 without any hinge structure. P1 has significantly decreased lytic activities against bacterial and tumor cells and PC/PS vesicles (3:1, w/w), and reduced pore-forming activity on lipid bilayers, while P2 retained effective lytic activities and pore-forming activity. The N-terminal region of P3 has a flexible structure without any specific secondary structure. The P3 modification caused a drastic decrease in the antibiotic activities, whereas P4, with the hydrophobic Leu side chain at position 2, retained its activities. On the basis of the tertiary structures, antibiotic activities, vesicle-disrupting activities, and pore-forming activities, the structure-function relationships can be summarized as follows. The partial insertion of the Trp2 of CA-MA into the membrane, as well as the electrostatic interactions between the positively charged Lys residues at the N-terminus of the CA-MA and the anionic phospholipid headgroups, leads to the primary binding to the cell membrane. Then, the flexibility or bending potential induced by the Gly-Ile-Gly hinge sequence or the Pro residue in the central part of the peptides may allow the alpha-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA-MA.     

Expression of insulin-like growth factor-1 of orange-spotted grouper (Epinephelus coioides) in yeast Pichia pastoris

IGF-1 plays a key role in development, growth, and metabolism in teleost. Recombinant fish IGF-1 may be a useful tool for both theoretical research and aquaculture applications. However, using the Escherichia coli expression system has several drawbacks for producing quality fish IGF-1 protein. To explore the yeast expression system for generating fish IGF-1 protein, the cDNA coding for the mature orange-spotted grouper IGF-1 peptide without signal peptide and E domain was cloned into the secreting expression organism Pichia pastoris. Tricine-SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rgIGF-1 was secreted into the culture medium, had a molecular weight of 8.7 kDa. The production peaked at 24h of induction and the optimal pH for expression was 5.0. The recombinant protein was purified using a combined ammonium sulfate precipitation with Ni(2+) affinity chromatography. Finally, 17.9 mg of the protein was obtained from 420 ml of the culture supernatant and the purity was about 92.4%. Bioactivity of the rgIGF-1 was confirmed by the ability to stimulate proliferation of embryo cell line of grouper (GP cell line) and MFC-7 cell. The present results suggest that the Pichia pastoris expression system can be used to produce a functional rgIGF-1 for both research and aquaculture application.     

Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells

A 20-residue hybrid peptide (CA(1-8)-MA(1-12): KWKLFKKIGIGKFLHSAKKF-NH(2)) incorporating 1-8 residues of cecropin A (CA) and 1-12 residues of magainin 2 (MA) has potent antibiotic activity without hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly of CA(1-8)-MA(1-12) (CA-MA) on antibiotic activity, CA-MA and its three analogues, CA-MA1, CA-MA2 and CA-MA3 were synthesized. The Gly-Ile-Gly sequence of CA-MA was deleted in CA-MA1 and replaced with Pro and Gly-Pro-Gly in CA-MA2 and CA-MA3, respectively. CA-MA1 and CA-MA3 caused a significant decrease in the bactericidal rate against Escherichia coli and Bacillus subtilis and the tumoricidal activity against four different tumor cells, and the PC/PS (4:1, w/w) vesicle-aggregating and disrupting activities. However, CA-MA2 showed a similar bactericidal rate and antitumor, vesicle-aggregating and disrupting activities, as compared with CA-MA. These results suggested that the flexibility or beta-turn induced by Gly-Ile-Gly or Pro in the central part of CA-MA may be important in the electrostatic interaction of the cationic short alpha-helical region in the N-terminus with the cell membrane surface and the hydrophobic interaction of amphipathic alpha-helical region in the C-terminus with the hydrophobic acyl chains in the cell membrane. CA-MA3 exhibited lower activity in antibacterial, antitumor, and vesicle-aggregating and disrupting activities than CA-MA and CA-MA2. This result suggested that the excessive beta-turn structure by Gly-Pro-Gly in CA-MA3 seems to interrupt the ion channel/pore formation on the lipid bilayer. It was concluded that the appropriate flexibility or beta-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.     

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.     

石斑鱼-防御素的酵母表达及其产物抗菌活性分析

防御素是一类阳离子抗菌肽。研究从石斑鱼垂体SMART cDNA 文库中扩增出129 bp 石斑鱼-防御素成熟肽序列, 将其克隆到毕赤酵母表达载体pPCIZA 中, 构建了石斑鱼-防御素的真核表达载体, 电击转化毕赤酵母GS115。Western Blot 分析表明石斑鱼-防御素在酵母菌中获得了表达。体外抗菌实验表明纯化的重组蛋白具有抑制大肠杆菌以及嗜水气单胞菌的作用, 但是对革兰氏阳性菌, 如金黄色葡萄球菌和藤黄微球菌的生长没有抑制作用。实验结果表明酵母表达的石斑鱼-防御素能够特异地抑制革兰氏阴性菌的生长。       

Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris--importance of correct processing of the N-terminus

This study was conducted to optimize the expression of human CB2 cannabinoid receptors in methylotrophic yeast Pichia pastoris (P. pastoris). Two major species of expressed CB2 proteins were seen on Western blot, i.e., a 42 kDa band which matches the calculated molecular weight for tagged CB2, and a 52/55 kDa doublet. Treatment of membranes with N-glycosidase F or inclusion of tunicamycin in the culture medium during induction resulted in the disappearance of the 55 kDa, but not the 52 kDa band, suggesting that the 3 kDa extra in the 55 kDa band is due to N-glycosylation, but the 10 kDa extra in the 52 kDa band is not due to N-glycosylation. Anti-FLAG M1 antibody had a much higher preference for the 42 kDa band over the 52/55 kDa doublet, and a 10 kDa fragment recognized by anti-FLAG M2 antibody was generated by CNBr digestion of the 52/55 doublet. These data strongly support the hypothesis that the 10 kDa increase in molecular weight was due to unprocessed alpha-factor sequence. This conclusion was further validated by finding several peptide sequences for alpha-factor fragments at the N-terminal of the CB2 receptor using pepsin/chymotrypsin digestion and LC/MS/MS approaches. Importantly, unprocessed alpha-factor was found to be associated with poor ligand binding. In addition, controlling the level of CB2 protein expression was found to be critical for minimizing the presence of unprocessed alpha-factor sequence. The information gained from this study should aid the proper expression of not only CB2 receptor but also other members of the GPCR family in P. pastoris.     

High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris

The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

杂合抗菌肽CecA-mil的改造及在毕赤酵母中的分泌表达

参照毕赤氏巴斯德酵母(Pichia pastorts)偏好密码子,改造并化学合成杂合抗菌肽CecA-mil基因,改造后的CecA-mil基因克隆到pPICZα-A载体中,构建分泌型重组酵母表达载体pPICZα-A-CM,转化Pichia pastoris受体菌X-33。在醇氧化酶(AOX)启动子调控下,分子量约1．9kD的CecA-mil杂合抗菌肽获得表达,经表达条件优化,重组酵母菌的摇瓶发酵产率可达到245μg／mL。抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G^-菌及G^ 菌均有较好的抑菌活性,特别是对氨苄青霉素抗性菌和卡那霉素抗性菌抑杀效果更好;具有热稳定性和酸稳定性。这些特点使得重组抗菌肽CecA-mil在食品防腐、疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity

Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate a synthetic antimicrobial peptide cp gene, which was then cloned into the expression vector pPICZalpha-A. The constructed recombinant expression vector pPICZalpha-cp was transformed into Pichia pastoris X-33, in which the synthetic antimicrobial peptide (CP) could be expressed under the control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Results showed that recombinant plasmid is highly stable, and In vitro experiments showed that the recombinant antimicrobial peptide CP is heat and acid-stable, and it has high antibacterial activity against several Gram-positive and -negative bacteria. Only 1 microg of the recombinant antimicrobial peptide CP has an antibacterial activity equivalent to 64 U ampicillin. Thus, this recombinant antimicrobial peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein.     

A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.     

一种新融合抗菌肽Hex-Mag基因的克隆、表达及其抗菌活性的研究

通过在Magainin1-12(GIGKFLHSAKKF)的N端添加碱性氨基酸片段Hexapeptide(RRWQWR)以增强其对细胞膜的吸附能力来提高Magainin1-12的抗菌活性。利用Hexapeptide和Magainin1-12的基因序列,结合酵母偏爱密码子设计出新的融合基因Hex-Mag,通过重叠区扩增基因拼接法(Gene splicing by overlap extension,gene SOEing)利用PCR扩增出基因片段,再将融合基因进行酶切并纯化后导入穿梭质粒pPIC9中,构建受乙醇氧化酶1基因(AOX1)的启动子与转录终止区控制的酵母表达质粒,转化GS115毕赤酵母宿主菌,经表型筛选,阳性克隆用甲醇诱导表达。表达出的融合肽Hex-Mag分子量约2.3kDa,其耐热性强,在100℃条件下,其活性可维持3h以上。琼脂糖孔穴扩散法检测显示Hex-Mag对多种革兰氏阴性菌和阳性菌具有抑制活性,与Magainin1-12相比,其活性有明显增强,N端正电荷增加的预期效应得到初步体现。     

杂合抗菌肽在毕赤酵母中的表达及其活性测定

为获得溶血活性低、抗菌活性高的杂合抗菌肽,以家蝇抗菌肽Cec Md和中国林蛙抗菌肽Chensirin为母体肽,并结合毕赤酵母偏爱密码子的原则,设计出6条具有抗菌潜力的新型杂合抗菌肽,将其命名为CC22、CC28、CC29、CC30和CC34(1),CC34,利用SOE-PCR技术合成所需的目的基因,并将其克隆至毕赤酵母表达载体pGAPZαA,通过电击转化技术,将其转化至毕赤酵母SMD1168中,经含有Zeocin的抗性平板筛选阳性转化子,YPD液体培养72h后,经Tricine-SDS-PAGE检测出目的蛋白,然后采用高效液相色谱法对其进行纯化。检测结果显示,表达产物CC29对大肠杆菌、鸡沙门氏菌的最小抑菌浓度(MIC)均为25μg/ml;CC34(1)对大肠杆菌表现相对较弱的抑制作用,最小抑菌浓度为100μg/ml;CC34对鸡沙门氏菌和金黄色葡萄球菌的最小抑菌浓度为50μg/ml;且杂合抗菌肽对有益菌均没有表现出抑制作用。6条杂合肽的溶血活性均呈现较低水平,其中表现出抗菌活性的3条抗菌肽中,以CC29的溶血活性最低,CC34(1)和CC34相对次之。结合抑菌活性,CC29和CC34的抑菌效果较为明显,从而确定溶血活性低且抗菌活性较高的CC29和CC34为新型杂合抗菌肽。     

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43kDa and 66.2kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60h of induction during culture, it was found that the degradation product of around 66.2kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).     

Recombinant expression of human cathelicidin (hCAP18/LL-37) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.