A truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase with improved enzymatic activity and thermotolerance

As an approach to improving Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase (Fsbeta-glucanase) for use in industry and to studying the structure-function relationship of the C-terminus in the enzyme, a C-terminally truncated ( approximately 10 kDa) Fsbeta-glucanase was generated using a PCR-based gene truncation method and then overexpressed in either Escherichia coli BL21(DE3) or Pichia pastoris strain X-33 host cells. The initial rate kinetics, protein folding, and thermostability of the wild-type and truncated glucanases were characterized. The truncated enzyme expressed in Pichia cells was found to be glycosylated and composed of two dominant polypeptide bands as judged by SDS-PAGE. An approximate 3-4-fold increase in the turnover rate (k(cat)), relative to that of the full-length enzyme, was detected for the purified truncated glucanases produced in E. coli (designated TF-glucanase) or Pichia host cells (designated glycosylated TF-glucanase). The glycosylated TF-glucanase is the most active known 1,3-1,4-beta-d-glucanase, with a specific activity of 10 800 +/- 200 units/mg. Similar binding affinities for lichenan (K(m) = 2.5-2.89 mg/mL) were detected for the full-length enzyme, TF-glucanase, and glycosylated TF-glucanase. Both forms of truncated glucanase retained more than 80% of their original enzymatic activity after a 10 min incubation at 90 degrees C, whereas the full-length enzyme possessed only 30% of its original enzymatic activity after the same treatment. This report demonstrates that deletion of the C-terminal region ( approximately 10 kDa) in Fsbeta-glucanase, consisting of serine-rich repeats and a basic terminal domain rich in positively charged amino acids, significantly increases the catalytic efficiency and thermotolerance of the enzyme.     

具血型转换功能的咖啡α-半乳糖苷酶基因的克隆与表达

从大粒种咖啡(Coffea liberica)和中粒种咖啡(Coffea canephora)中分离克隆到了α-半乳糖苷酶（α-D-galactosidase）cDNA的开放阅读框架即编码区,分别记为Gal-D与Gal-Z,长度与已发表的小粒种咖啡cDNA编码序列相同均为1 089 bp,同源性与已发表的小粒种咖啡cDNA编码序列相比分别为98.7%和99.27%。 将克隆到的Gal-D与Gal-Z用巴斯德毕赤氏酵母Pichia pastoris表达载体pPICZαA（分泌甲醇诱导型）和pGAPZαA（分泌组成型）成功地构建了如下酵母表达载体：pPICZαA/Gal-Z,pPICZαA/Gal-D和pGAPZαA/Gal-D,并转入酵母宿主菌GS115进行了发酵表达研究。实验得出工程菌株pPICZαA/Gal-D / GS115的重组表达产物酶活最高可达48.22（U/mL）,对发酵产物进行了SDS-PAGE电泳,得到一条清晰的主条带。     

里氏木霉纤维二糖水解酶Ⅱ在毕赤酵母中的高效表达

本工作采用巴氏毕赤酵母Pichiapastoris表达系统进行了里氏木霉Trichodermareesei纤维二糖水解酶Ⅱ(CellobiohydrolaseII)的表达。用RT-PCR的方法从经稻草粉诱导的里氏木霉培养物中分离出纤维二糖水解酶Ⅱ的基因,将其插入到巴氏毕赤酵母的表达载体pPICZαA中,并使之处于α-因子信号肽序列的下游,得到重组质粒pPICZαA-cbh2。通过电穿孔的方法用线性化的pPICZαA-cbh2转化巴氏毕赤酵母GS115菌株,经过大量筛选后得到可以高效表达纤维二糖水解酶的毕赤酵母菌株P.pastorisCBHⅡ1。在甲醇诱导的条件下培养P.pastorisCBHⅡ1,培养液中的CMC活性可达到3.82U/mL,SDS-PAGE分析结果表明纤维二糖水解酶在P.pastorisCBHⅡ1中的表达量远远高于里氏木霉。对表达产物进行了LC-MS分析,结果表明所表达的蛋白为里氏木霉的纤维二糖水解酶。     

绿色木霉LTR-2 β-1, 3-葡聚糖酶基因的克隆及其在 毕赤酵母中的表达

根据从GenBank中检索到的木霉菌β-1,3-葡聚糖酶基因序列设计引物,以高产β-1,3-葡聚糖酶菌株--绿色木霉LTR-2的cDNA为模板,采用PCR方法扩增得到内切β-1,3-葡聚糖酶基因(glu).将glu克隆至载体pMD18-T上,进行了全序列测定.序列分析表明该基因由2289个核苷酸残基组成,含有一个开放阅读框架,可以编码762个氨基酸,与报道基本相同.翻译后的氨基酸序列含有两个β-1,3-葡聚糖酶的保守区RVVYIPPGTY和AASQNKVAYF.基因与已发表的木霉β-1,3-葡聚糖酶基因有较高的同源性,其中和哈茨木霉bgn3.1和绿木霉bgn13.1的同源性达到93%.序列已经提交GenBank,登录号为EF176582.将glu基因插入到巴斯德毕赤酵母(Pichia pastoris)穿梭载体pPIC9K中,获得重组质粒pGLU14,经线性化后转化毕赤酵母菌株KM71.经大量平板筛选,获得能有效分泌表达β-1,3-葡聚糖酶的毕赤酵母工程菌株KGLU14,菌落PCR扩增证实了glu基因已经整合到酵母基因组中.SDS电泳结果表明其β-1,3-葡聚糖酶的分子量大约为80kDa,和理论推测值大致相同.摇瓶发酵结果表明,培养基中β-1,3-葡聚糖酶的活力可达889U/mL.     

High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.     

Synthesis of alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on human tumor cells by recombinant alpha1,3galactosyltransferase produced in Pichia pastoris.

This study describes the processing of human tumor cells or cell membranes to express alpha-gal epitopes (Galalpha1-3Gal-beta1-4GlcNAc-R) by the use of New World monkey (marmoset) recombinant alpha1,3galactosyltransferase (ralpha1,3GT), produced in the yeast Pichia pastoris. Such tumor cells and membranes may serve, in cancer patients, as autologous tumor vaccines that are targeted in vivo to antigen-presenting cells by the anti-Gal antibody. This ralpha1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solubility without detergent. It is effectively produced in P. pastoris under constitutive expression of the P(GAP) promoter and is secreted into the culture medium in a soluble, truncated form fused to a (His)(6) tag. This tag enables the simple affinity purification of ralpha1,3GT on a nickel-Sepharose column and elution with imidazole. The purified enzyme appears in SDS-PAGE as two bands with the size of 40 and 41 kDa and displays the same acceptor specificity as the mammalian native enzyme. ralpha1,3GT is very effective in synthesizing alpha-gal epitopes on membrane-bound carbohydrate chains and displays a specific activity of 1.2 nM membrane bound alpha-gal epitopes/min/mg. Incubation of very large amounts of human acute myeloid leukemia cells (1 x 10(9 )cells) with neuraminidase, ralpha1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 10(6 )alpha-gal epitopes per cell. Effective synthesis of alpha-gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcinoma. These data imply that ralpha1,3GT produced in P. pastoris is suitable for the synthesis of alpha-gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.     

用GAP启动子在Pichia pastoris GS115中组成型表达鼠灰链霉菌腺苷酸脱氨酶

【目的】构建产AMP脱氨酶的重组毕赤酵母(Pichia pastoris GS115)菌株,并初步优化其发酵条件。【方法】以鼠灰链霉菌(Streptomyces murinus)基因组为模板PCR扩增获得腺苷酸脱氨酶基因AMPD,以pGAP9K为载体构建重组表达质粒pGAP9K-AMPD并通过电转化法转入Pichia pastoris GS115,筛选转化子对其酶活进行测定,并初步优化其发酵条件。【结果】构建了毕赤酵母重组菌,通过分光光度法测定,显示重组菌有明显的酶活;初步优化发酵条件为:该重组菌最适发酵培养基为:甘油2%,蛋白胨2%,酵母膏1%,KH2PO40.5%,MgSO4·7H2O0.05%,pH 6.0;发酵条件为:接种龄24 h,转接量3%,30°C﹑200 r/min培养96 h,取发酵上清液测定酶活,重组菌腺苷酸脱氨酶酶活达到2 230±60 U/mL。【结论】构建了一株产AMP脱氨酶活性较高的重组毕赤酵母菌株,并通过优化发酵条件使其酶活达到2 230±60 U/mL。为AMP脱氨酶工业化生产奠定了一定的基础。     

重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

目的:为了获得有催化活性的人乙酰半乳糖胺转移酶3(GALNT3),构建了GALNT3可溶性区域(GALNT3-sol)的真核分泌表达载体,在巴斯德毕赤酵母中表达并纯化GALNT3-sol蛋白,体外检测其转糖基活性。方法:以构建好的pET15b/GALNT3-sol为模板进行PCR,扩增编码人GALNT3-sol的cDNA片段(1 755 bp),将其克隆至真核表达载体pPIC9K,载体线性化后采用电击法转化毕赤酵母GS115。通过MD平板和G418平板筛选出阳性高拷贝重组菌株。阳性菌株经过甲醇诱导表达人GALNT3-sol重组蛋白,表达上清进行Ni-NAT分离纯化。分别采用SDS-PAGE和Western blot鉴定纯化的重组蛋白,并使用HPLC和MALDI-TOF/MS分析其转糖基化反应的活性。结果:成功构建了能够分泌表达GALNT3-sol的毕赤酵母菌株。阳性表达菌株在BMMY培养基(pH 6.0)中20℃培养,经0.5%甲醇诱导表达96 h,摇瓶表达量可达5mg/L。SDS-PAGE和Western blot结果显示表达重组蛋白为糖基化形式。活性检测显示表达的重组蛋白具有转糖基活性。结论:成功获得可以高效分泌表达具有活性的人GALNT3-sol蛋白的毕赤酵母菌株,为进一步研究人GALNT3的性质及其应用提供了基础。     

Expression of human gelatinase B in Pichia pastoris.

Full-length human gelatinase B (FLGelB) and its C-terminal truncated form (dGelB) were expressed in Pichia pastoris strain GS115, using the Saccharomyces cerevisiae Mat alpha signal peptide. In both cases, a high level of the secreted protein could be detected by SDS-PAGE. The truncated gene was also expressed using the human gelatinase B native signal peptide. Secretion using the Mat alpha signal peptide was significantly greater than that from the native signal peptide. The recombinant products were purified and characterized biochemically. The recombinant proteins, FLGelB and dGelB, were found to have similar biochemical properties and activity to that of the human gelatinase B native protein.     

重组尿激酶型纤溶酶原激活物受体在酵母系统中的表达及鉴定

构建截断型可溶性尿激酶型纤溶酶原激活物受体（ｕ－ＰＡＲ）的原核及酵母表达质粒并分别在大肠杆菌和Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母中高效表达．利用ＰＣＲ扩增截断型可溶性ｕ－ＰＡＲｃＤＮＡ片段,并分别连入酵母及原核表达载体中,构建成表达质粒．后者经诱导表达的蛋白被用于免疫家兔,获得抗ｕ－ＰＡＲ的抗血清,用于对酵母表达产物的鉴定．前者在甲醇的诱导下表达,产物分泌至培养液中．结果表明,原核表达的ｕ－ＰＡＲ蛋白免疫家兔后获得高效价的抗血清,利用此抗血清所作Ｗｅｓｔｅｒｎｂｌｏｔ证实酵母表达蛋白具有ｕ－ＰＡＲ的免疫原性,表观分子量４０ｋＤ左右．Ｍｕｔ－表型在第５ｄ为最高（２．５μｇ／ｍｌ）,Ｍｕｔ＋表型在第４ｄ为最高（７．５μｇ／ｍｌ）．因此,构建的可溶性ｕ－ＰＡＲ表达质粒在Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母细胞获得表达,为进一步竞争拮抗受体功能的研究奠定了基础．     

An ultra scale-down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges

Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra‐scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot‐scale disk‐type machines, was used in this work. Two recombinant P. pastoris strains, namely a X‐33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot‐ and a large‐scale disk‐type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X‐33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot‐scale runs during early stages of process development for therapeutic products. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1029–1036, 2012     

High-Level Production and Purification of P30P2MSP119, an Important Vaccine Antigen for Malaria, Expressed in the Methylotropic Yeast Pichia pastoris

P30P2MSP1(19) is a recombinant subunit vaccine derived from merozoite surface protein 1 (MSP1) of Plasmodium falciparum, the causative agent of malaria. P30P2MSP1(19) consists of two universal T-cell epitopes fused to the most C-terminal 19-kDa portion of MSP1, and this protein has previously shown promising potential as a vaccine for malaria. However, previous attempts at producing this molecule in Saccharomyces cerevisiae resulted in the production of a truncated form of the molecule missing most of the universal T-cell epitopes. Here, we report the production of full-length P30P2MSP1(19) in Pichia pastoris. As salt precipitation is a common problem during P. pastoris high-density fermentation, we utilized an alternative low-salt, fully defined medium that did not reduce growth rates or biomass yields to avoid precipitation. A total of 500 mg/L of secreted purified protein was produced in high cell density fermentation and the protein was purified in one step utilizing nickel-chelate chromatography. P30P2MSP1(19) produced in Pichia was reactive with monoclonal antibodies that recognize only conformational epitopes on correctly folded MSP1. Rabbits immunized with this molecule generated higher and more uniform antibody titers than rabbits immunized with the protein produced in Saccharomyces. P30P2MSP1(19) produced in Pichia may prove to be a more efficacious vaccine than that produced in Saccharomyces and Pichia would provide a system for the cost-effective production of such a vaccine.     

Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone

Human parathyroid hormone (hPTH) is involved in calcium metabolism, and the unique ability of this hormone to stimulate bone growth makes it a promising agent in the treatment of osteoporosis. We have engineered the methylotrophic yeast Pichia pastoris for the production of over 300 mg intact hPTH per liter growth medium. The presence of 10 mM EDTA in the culture medium was essential to obtain this high hormone yield, indicating that metallopeptidases are mainly responsible for the otherwise instability of hPTH. Furthermore, the secretion process of hPTH was considerably improved by coexpression of Saccharomyces cerevisiae protein disulphide isomerase (ScPDI). Since hPTH does not contain any cystein residues, this effect may be indirect or ascribed to the chaperone activity of PDI. Contrary to the situation in S. cerevisiae, use of a protease-deficient host strain provided no additional advantage. The hormone secreted by P. pastoris was not subjected to proteolytic processing by Kex2p in the two internal tribasic sites, nor were any C-terminal truncated hPTH forms detected. However, the P. pastoris hPTH producing transformants cosecreted ubiquitin to the culture medium, possibly as a result of a stress-related response.     

Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell- enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.      

猪干扰素-γ基因在毕赤酵母中的分泌表达

将去除信号肽的猪干扰素-γ（PoIFN-γ）基因置于酿酒酵母α因子分泌信号的DNA序列后, 构建成pPIC9K-α-PoIFN-γ分泌型重组表达载体, 电转化导入毕赤酵母GS115中,经G418筛选后获得2株多拷贝插入的重组子。SDS-PAGE和Western blot分析结果表明,所获得的重组子能够分泌表达出17kD和23kD左右的PoIFN-γ特异蛋白,其表达量为108mg/L,占培养液总蛋白的60%。实验首次在毕赤酵母表达系统中实现了PoIFN-γ基因的分泌表达。     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

Production of alpha-galactosyl epitopes via combined use of two recombinant whole cells harboring UDP-galactose 4-epimerase and alpha-1,3-galactosyltransferase

alpha-Galactosyl epitopes (or alpha-Gal, oligosaccharides with a terminal Galalpha1,3Gal sequence) are a class of biologically important oligosaccharides in great demand in bulk quantities for basic and clinical studies on preventing hyperacute rejection in pig-to-primate organ xenotransplantation. A truncated bovine alpha-1, 3-galactosyltransferase, the key enzyme responsible for the biosynthesis of the terminal structure of alpha-Gal, was cloned and overexpressed previously. The acceptor specificity was further studied in the present paper, and lactose and galactose derivatives were found to be good acceptors. To develop a more proficient reaction process, we report herein an example of an efficient enzymatic synthesis of alpha-Gal oligosaccharides catalyzed by the combination of two recombinant Escherichia coli whole cells harboring the genes of a UDP-galactose 4-epimerase and the alpha-1, 3-galactosyltransferase, respectively. Using lactosyl azide (LacN(3)) as the acceptor for the glycosyltransferase, the combined use of the two recombinant cells efficiently produced alpha-Gal epitope Gal alpha1,3LacN(3) in 60-68% yield.     

毕赤酵母液滴微流控高通量筛选方法的建立与应用

巴斯德毕赤酵母是当前应用最为方便和广泛的外源蛋白表达系统之一,为了进一步提高其表达外源蛋白的能力,文中建立了基于液滴微流控的毕赤酵母高通量筛选方法,并以木聚糖酶融合荧光蛋白为例,筛选获得木聚糖酶表达和分泌能力提高的突变株。通过PCR扩增得到木聚糖酶xyn5基因和绿色荧光蛋白gfp基因融合片段,并克隆到毕赤酵母表达载体pPIC9K中构建出木聚糖酶融合绿色荧光蛋白的质粒pPIC9K-xyn5-gfp,电转化至毕赤酵母GS115中得到表达木聚糖酶和绿色荧光蛋白的毕赤酵母SG菌株。该菌株经过常压室温等离子体诱变后进行单细胞液滴包埋,液滴培养24h后进行微流控筛选,获得高表达木聚糖酶的突变菌株,进而用于下一轮的诱变突变库构建和筛选。以此类推,经过5轮液滴微流控筛选,获得一株高产菌株SG-m5,其木聚糖酶活为149.17U/mg,较出发菌株提升300%,分泌外源蛋白的能力较出发菌株提高160%。文中建立的毕赤酵母单细胞液滴微流控高通量筛选方法能达到每小时10万菌株的筛选通量,筛选百万级别的菌株库仅需10h,消耗荧光试剂体积100μL,对比传统的微孔板筛选方法降低试剂成本近百万倍,为高效、低成本筛选获得表达和分泌外源蛋白能力提高的毕赤酵母提供了一条新途径。     

In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.