Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression

A new method for the isolation of glucose repression-insensitive mutants in the methylotrophic yeast Pichia pinus was developed. The method is based on screening of small suspension samples derived from 2-deoxyglucose-resistant colonies for alcohol oxidase activity. Alcohol oxidase activity was evaluated by determination of formaldehyde excreted by cells. Mutants with glucose non-repressible alcohol oxidase and catalase synthesis were obtained. All mutants grew poorly on D -xylose compared to the wild type, whereas growth on L -arabinose was similar to the wild type. Changes in the glucose transport system were suggested to be responsible for altered growth characteristics and defective glucose repression.     

白细胞介素-10基因多拷贝表达盒的构建及在毕赤酵母中的表达

目的构建白细胞介素-10(IL-10)基因多拷贝表达盒,提高IL-10在毕赤酵母中的表达水平。方法体外构建重组表达载体αIL-10/pAO815,BamHⅠ和BglⅡ双酶切获得目的基因表达盒(AOX-αIL-10),再连接到BglⅡ酶切位点处,依次构建多拷贝重组载体n(AOX-αIL-10)/pAO815。从质粒pPIC9K上用NdeⅠ和SalⅠ双酶切获得Kan抗性基因,重组到多拷贝表达载体n(AOX-αIL-10)/pAO815上。重组载体n(AOX-αIL-10)/pAO815电转化毕赤酵母,PCR筛选含有IL-10基因的酵母转化子,甲醇诱导表达,对表达量高的转化子再次经重组载体n(AOX-αIL-10)/pAO815-Kan电转化,高浓度G418抗性筛选二次酵母转化子,使用甲醇诱导表达。ELISA测定IL-10含量,MC/9细胞测定IL-10活性。结果所构建的8拷贝表达盒的重组载体8(AOX-αIL-10)/pAO815和4拷贝表达盒4(AOX-αIL-10)/pAO815-Kan,转化子分泌表达IL-10水平最高,为(8.25±1.65)mg/L,比活性为1.465×105U/mg。结论已成功构建了高拷贝表达盒,并提高了IL-10在毕赤酵母中的表达水平。     

两株抗氧化活性酵母的鉴定及对乳成分的影响

为获得有抗氧化活性的工业用酵母菌株,对2株来源于原料乳中有较强抗氧化活性的酵母菌进行鉴定.根据菌株的表型、生理生化特征和基因型的特性,初步将这2株菌鉴定为毕赤氏发酵酵母(Pichia fermentans).将这2株酵母菌在高温灭菌(UHT)牛乳中培养以研究其对牛乳主要成分的影响,结果表明:这2株酵母能在UHT牛乳中产乙醇,具有促进蛋白水解活性与脂肪水解活性的能力,能有效提高乳中的游离氨基酸和脂肪酸的含量.     

乳源酵母Pichia fermentans的抗氧化特性

为得到新的天然抗氧化剂,采用DPPH法和抑制脂质过氧化法对11株来源于牛乳的酵母菌抗氧化活性进行测定,包括他们的完整细胞和无细胞提取物,发现Pichia fermentans BY5有较好的抗氧化活性.分析不同条件下P.fermentans BY5细胞提取物的抗氧化活性,发现培养基的起始pH值、培养时间、培养基种类对酵母菌的抗氧化活性和蛋白质含量影响显著(P0.05);而抗氧化活性与蛋白质含量显著相关(P0.01).将酵母BY5在pH6的麦芽培养基中培养2d后清除DPPH活性可达60%;在pH6的YPD培养基中培养4d后抑制脂质过氧化能力达到74%.     

HBsAg/GM-CSF融合蛋白的克隆及其在毕赤酵母中的表达

目的在毕赤酵母中表达HBsAg/GM-CSF融合蛋白。方法利用PCR扩增HBsAg和GM-CSF基因,通过15个氨基酸的连接肽将两个片段连接,获得融合基因S-GM,克隆入酵母穿梭质粒pPIC9K中。将重组质粒9K-S-GM电转化毕赤酵母后,G418筛选,甲醇诱导,HBsAg/GM-CSF融合蛋白表达。经SDS-PAGE检测表达水平,Western blot检测表达产物特异性。结果PCR扩增的片段与预期大小一致,HBsAg/GM-CSF融合蛋白在毕赤酵母中获得了表达。Western blot检测,该融合蛋白同时具有HBsAg和GM-CSF的特异性。结论该融合蛋白的获得为提高乙肝疫苗的免疫原性奠定了科学基础。     

过敏原肌质钙结合蛋白在毕赤酵母中的表达、优化及免疫反应性研究

目的 探究天然肌质钙结合蛋白(sarcoplasmic calcium binding protein, SCP)的可替代物,为蟹类过敏原的检测提供基础材料,本研究首次利用毕赤酵母(Pichia pastoris, P. pastoris)高效表达表达三疣梭子蟹(Portunus trituberculatus)重要过敏原SCP,并检验其免疫反应性。方法 根据毕赤酵母的密码子偏好性优化SCP基因并构建重组质粒。将其热激转化至P. pastoris GS115菌株后经遗传霉素(Geneticin, G418)筛选获得阳性高拷贝子。最后通过甲醇诱导表达重组SCP并结合免疫印记(Western blotting, WB)和间接酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)验证其免疫反应性。结果 SCP在P. pastoris GS115中实现了可溶性高效表达,其表观分子量约为28 kDa。在摇瓶水平下,最佳诱导条件为pH为6.0、每24 h添加1.0%(v/v)甲醇,于28℃发酵144 h,在此条件下,纯度为91.6%的SCP产量可达15 mg/L。WB和间接ELISA结果表明,重组SCP具有IgG结合能力。结论 毕赤酵母表达系统可以得到纯度较高且免疫反应性良好的重组SCP。本研究为SCP的理化研究及产业化应用奠定了基础,并有望促进特异性甲壳类过敏原检测的发展。     

季也蒙毕赤酵母Y35-1菌株对枇杷采后炭疽病的抑菌效果及保鲜作用

利用从原生态枇杷果实上分离筛选并鉴定的、能显著抑制炭疽病的季也蒙毕赤酵母Y35-1菌株,研究其抑菌抗腐效果,并将其制成液体和固体型菌剂在枇杷贮藏保鲜方面应用。结果表明：拮抗酵母Y35-1菌株能够有效抑制枇杷炭疽病胶孢炭疽菌孢子的萌发,能够在病原菌菌丝上寄生而抑制菌丝的正常生长,不接触对峙培养实验显示Y35-1菌株能够有效抑制病原菌菌丝的扩展和生长。保鲜应用表明：酵母Y35-1液体和活性冻干粉菌剂均能明显抑制枇杷果实贮藏期间的病害腐烂发生率,贮藏至第20天时,两处理组果实腐烂指数分别仅为2.25%和3.60%;且这两种生防菌剂对枇杷的硬度、质量损失率、可溶性固形物、果皮细胞膜透性、总糖含量、总酸含量、VC含量均起到了一定的维持作用。Y35-1酵母液体菌剂在4?℃条件下、活性冻干粉菌剂在－20?℃条件下最适宜存放,且海藻糖对Y35-1酵母贮藏期间生活力和生防效力起到了保护作用。研究结果显示Y35-1菌株对采后枇杷胶孢炭疽菌有明显的抑菌作用,液体和固体剂型的生防制剂均对采后枇杷具有抑腐保鲜效果。     

重组毕氏酵母表达rhs-TRAIL发酵条件的优化研究

为了提高重组人可溶性肿瘤坏死因子相关的凋亡诱导配体(rhs—TRAIL,114-281)在毕氏酵母工程菌中的表达量,采用1000mL摇瓶培养体系,优化研究了在不同培养基,pH值、表达时间、甲醇浓度等培养条件下的表达情况。经条件优化,最终确定了培养基为完全培养基BMGY／BMMY,甲醇浓度为0．5％,pH为6.0,表达时间为96h。在此优化条件下,目的蛋白表达量可以达到68mg/L。     

Expression and purification of goat lactoferrin from Pichia pastoris expression system

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.     

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.