人源性抗HBsAg Fab抗体的发酵生产研究

为了适应工业生产的需要,利用fed—batch方法,重组人源性抗HBsAg Fab抗体酵母工程菌在30L发酵罐中进行了高密度发酵,发酵最适温度30℃,pH值范围5．0～5．3,溶氧范围20％～30％。发酵液OD600值达到300时开始诱导,甲醇最佳诱导浓度为10mL／L。重组人源性抗HBsAg Fab抗体经离子交换层析纯化,纯化产品经SDS-PAGE、Western blot进行分析和ELISA方法进行活性测定。结果显示,重组Fab抗体在Fed-batch发酵系统中可高效表达,经过192h的发酵生产,重组人源性抗HBsAg Fab抗体的表达量可达412mg／L。发酵上清经过离子交换层析纯化,获得纯度为95％的重组Fab抗体,该Fab抗体经ELISA分析具有较高的HBsAg抗原亲和力和特异性。结果证实可以通过高密度发酵毕赤酵母工程菌来高效生产重组人源性抗HBsAg Fab抗体,为后续的工业化生产应用奠定了基础。     

人源性抗HBsAg抗体Fab段在酵母中的表达

通过分步整合的方式,将人源性抗乙肝表面抗原（HBsAg）抗体Fab的轻、重链基因分步整合到巴斯德毕赤（Pichia pastoris）酵母GS115菌株的染色体上,经甲醇诱导,成功地分泌表达出抗HBsAg抗体的Fab片段,表达量达50～80mg/L。ELISA结果显示重组酵母分泌表达出的Fab具有较强的结合HBsAg的能力。通过抗Fab的抗体柱亲和层析,纯化出了纯度较高的Fab产品。     

Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.

We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermentors. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degrees C), (2) combined use of two beta-lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermentors. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the crude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH(2)-CGS YNR GSF SQS SGLV-CONH(2) had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%.     

Expression, purification, and characterization of humanized anti-HBs Fab fragment

Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.     

Production of a recombinant antibody fragment in whole insect larvae

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress™) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress™ system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5

Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced. The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding. Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains. The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG. Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system.     

Recombinant anti-stefin A Fab fragment: sequence analysis of the variable region and expression in Escherichia coli

Human stefin A is an inhibitor of lysosomal cysteine proteinases cathepsin B, H, L and S. In the present report we describe the cloning and expression of anti-stefin A Fab fragment A22 in E. coli. We have determined the nucleotide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences. Expression of the two antibody chains was achieved using a single vector with a PhoA promoter and coding regions placed after the signal sequences, directing them to the periplasmic space. The A22 Fab fragment was extracted from the periplasmic space and expression was confirmed by Western blot analysis. The recombinant A22 Fab fragment had an affinity for stefin A comparable to the original monoclonal antibody, as determined by ELISA.     

Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II

L-Asparaginase (isozyme II) from Escherichia coli is an important therapeutic enzyme used in the treatment of leukemia. Extracellular expression of recombinant asparaginase was obtained by fusing the gene coding for asparaginase to an efficient pelB leader sequence and an N-terminal 6x histidine tag cloned under the T7lac promoter. Media composition and the induction strategy had a major influence on the specificity and efficiency of secretion of recombinant asparaginase. Induction of the cells with 0.1 mM IPTG at late log phase of growth in TB media resulted in fourfold higher extracellular activity in comparison to growing the cells in LB media followed by induction during the mid log phase. Using an optimized expression strategy a yield of 20,950 UI/L of recombinant asparaginase was obtained from the extracellular medium. The recombinant protein was purified from the culture supernatant in a single step using Ni-NTA affinity chromatography which gave an overall yield of 95 mg/L of purified protein, with a recovery of 86%. This is approximately 8-fold higher to the previously reported data in literature. The fluorescence spectra, analytical size exclusion chromatography, and the specific activity of the purified protein were observed to be similar to the native protein which demonstrated that the protein had folded properly and was present in its active tetramer form in the culture supernatant.     

抗VEGF单克隆抗体Fab片段在E.coli中的分泌表达

目的：在大肠杆菌中构建、表达和纯化抗血管内皮生长因子（VEGF）的Fab片段（兰尼单抗,ranibizumab）,通过发酵条件的控制实现其在大肠杆菌周质和胞外的高效分泌表达,并检测其抗VEGF的活性。方法：以pET30a为质粒载体,构建了Fd链和L链前都含有OmpA信号肽、SD序列和T7 promoter的克隆载体pET30a（+）-LC-HC,转化BL21（DE3）表达菌株,并进行了培养基、温度和IPTG诱导浓度的条件优化。结果：确定Fab片段在大肠杆菌分泌表达摇瓶发酵最佳条件为：在含有1.5% Tryptone,1% Yeast Extract,0.5% Glucose,0.15% NaCl,0.1% NH₄Cl,0.08% MgCl₂·6H₂O的1L培养基的摇瓶中,按照10%的接种量,37℃摇床培养至对数生长后期（OD₆₀₀为2左右）,添加0.1mmol/L IPTG诱导剂,于16℃条件下诱导表达过夜（16h左右）。用周质破菌提取分泌至大肠杆菌周质腔的Fab片段,同时用中空纤维柱浓缩发酵培养基,最后用ProteinG亲和层析柱一步纯化洗脱,经SDS-PAGE检测分析和Brandford法测蛋白浓度得出纯化的Fab抗体片段纯度在90%以上,分泌表达纯化量为0.4mg/L。以VEGF165作为结合抗原,间接ELISA分析纯化后的Fab抗体EC50=30ng/ml。继续用该培养基在3.7L体积发酵罐中进行2L体积的发酵,获得最终的菌体产率为30g/L,可亲和纯化Fab抗体量为1.94mg/L。结论：成功实现了Fab抗体片段在大肠杆菌中的高效分泌表达,且具有很高的活性,为规模化制备Fab抗体片段提供了研究依据。     

Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.     

High level expression of a promising anti-idiotypic antibody fragment vaccine against HIV-1 in Pichia pastoris

We have expressed the anti-idiotypic antibody 3H6 Fab directed against the HIV-1 broadly neutralising antibody 2F5 in methylotrophic yeast Pichia pastoris. The chimeric human/mouse Fab fragment was expressed under control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Bioreactor experiments showed the ability of the recombinant P. pastoris clone to secrete up to 260 mg/L Fab fragment in the culture supernatant during a five days cultivation time. Codon optimisation of the Fab expression cassette gave no further improvement of specific productivity when comparing 12 clones of each construct. The subsequent purification of Fab containing supernatants was done by anion exchange and size-exclusion chromatography with a recovery resulting in 70% of the recombinant protein. For verification of the suitability of the expression system we characterised the expressed protein with respect to both, its specificity and binding affinity and could not detect any significant difference between products from yeast derived and the hybridoma derived product. Finally we tested the implicit requirement of the carbohydrate moiety in the H2 loop of the original 3H6 antibody by introducing an asparagine to alanine replacement and, in a second experiment, inhibition of N-glycosylation by tunicamycin treatment. Biochemical analysis confirmed that the N-glycosylation does not contribute to the binding properties of 3H6.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

基因工程α-半乳糖苷酶的制备及其性质研究

在获得可分泌表达α 半乳糖苷酶基因工程毕赤酵母菌株的基础上 ,尝试了基因工程α 半乳糖苷酶在 5L发酵罐中的表达以及从发酵液中纯化α 半乳糖苷酶的研究。在 4L无机盐培养基中接种 0 .4LpPIC9K Gal GS115培养物 ,最终得到 3 .5L发酵液。离心所得上清中总蛋白含量为 2 .1g L。根据发酵液中目的蛋白含量高、杂质少等特点 ,设计了如下的纯化流程 :离心→超滤→阳离子交换层析→脱盐→浓缩。纯化后电泳银染结果呈单一蛋白带 ,总回收率 41%。通过测定米氏常数等生化性质对重组酶进行鉴定后 ,完成了人B型红细胞的酶解实验。结果表明 ,从发酵液中纯化的α 半乳糖苷酶可将B型红细胞改造成O型红细胞。本研究同时在数量和质量上为α 半乳糖苷酶在众多领域的广泛应用奠定了基础。     

Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter

The expression of a monoclonal antibody Fab fragment in Escherichia coli strain RB791/pComb3, induced with either lactose or isopropyl-beta-D-thiogalactoside (IPTG), was compared to determine if lactose might provide an inexpensive alternative to induction with IPTG. Induction of Fab expression imposed a metabolic load on the recombinant cells, resulting in lower final cell yields compared to the non-induced controls. An IPTG concentration of 0.05 mM was sufficient to achieve maximal expression of soluble Fab protein when inducing in the early-, mid-, or late-log phases of batch cultures grown using either glucose or glycerol as a carbon source. The largest overall yield of Fab fragments when using 0.05 mM IPTG was achieved by increasing the final yield of cells through glycerol feeding following induction in late-log phase. Lactose was as effective as IPTG for inducing Fab expression in E. coli RB791/pComb3. The greatest overall level of Fab expression was found when cells grown on glycerol were induced with 2 g/L lactose in late-log phase. Since the cost of 0.05 mM of IPTG is significantly greater than the cost of 2 g/L lactose, lactose provides an inexpensive alternative to IPTG for inducing the expression of Fab fragments, and possibly other recombinant proteins, from the E. coli lac promoter.     

血管抑制素功能结构域K1与PKA底物磷酸化基序的融合表达及磷酸化标记

通过 RT- PCR,从人肝组织中扩增出血管形成抑制素 ( angiostatin) c DNA的 K1片段 ,经DNA序列分析证实其正确性 ;将 K1与 GST融合并带上 1 7个氨基酸的 PKA底物磷酸化基序 ,IPTG诱导表达 ,以还原型谷胱甘肽偶联的琼脂糖凝胶亲合层析直接从细菌裂解上清中纯化融合蛋白 ;以 PKA催化单位将 3 2 P通过磷酸化作用标记至纯化的蛋白 ,再用凝血酶切去 GST,进行SDS- PAGE.放射自显影结果显示 ,GSTag- K1和 Tag- K1分别在 40 k D和 1 7k D处有信号强而特异的显影条带 ,表明带有磷酸化序列的蛋白能够被 PKA特异地磷酸化标记     

高密度发酵制备重组人OCIF活性域多肽融合蛋白

以补料-分批发酵方式在3．7L发酵罐上实现了人破骨细胞形成抑制因子活性域多肽(OCIF AD)的高密度发酵融合表达。通过参数控制。发酵液最终OD600达12．5(相当于35g湿菌体,L),表达量占菌体总蛋白40％左右,含量超过0．6g/L,并且90％,呈可溶性而直接具有生物学活性。直链淀粉树脂亲和层析法一步纯化,纯度达90％以上。     

Recognition of defined epitopes by affinity‐purified anti‐immunoglobulin Fab autoantibodies isolated from HIV‐infected humans

Infection of humans with HIV‐1 has previously been independently shown to result in the generation of autoantibodies (AAbs) reactive with immunoglobulin Fab fragments (Heidelberg), and with autoantibodies to T‐cell receptors (TCRs) (Tucson). Here, we carry out epitope mapping studies of affinity‐purified AAbs to Fab fragments prepared from individual HIV‐positive patients for their capacity to bind recombinant constructs and peptide‐defined epitopes modeling TCR and Ig light chains. Some affinity‐purified autoantibodies reacted strongly with TCRs expressed by intact T‐cells, and recombinant V_α/V_β constructs as well as with certain synthetic peptide epitopes. The binding reactions of affinity‐purified AAbs of individual patients were distinct, and the AAb preparations consisted of populations of polyclonal lgs as reflected in specificity and isotype. AAb pools from individual patients all bound particular regions of TCR and Ig chains defined by comprehensive peptide synthesis including the CDR1 and Fr3 segments of the variable domains and the joining segment/switch peptide. In addition, other reactivities to restricted regions of α, β and λ light chains were documented. These results substantiate the cross‐reactivity of TCR and Ig–Fab determinants, and are consistent with the hypothesis that autoantibodies arising as a consequence of HIV infection can have an immunomodulatory role. Copyright © 1999 John Wiley & Sons, Ltd.     

Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives

Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs. We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains. Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated. Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed. To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography.