Versatile expression system for rapid and stable production of recombinant proteins

Previously we reported the development of a novel expression system with Tat/TAR-oriP vectors and HKB11 cell line, which supports high level protein expression (Cho et al. Cytotechnology 2001, 37, 23-30). In the present study, we further demonstrated that HKB11 cells are suitable for high throughput expression (microgram scale) of genomic candidates in transient transfection system for in vitro evaluation of biological functions. HKB11 cells were also shown to support the production of milligram to gram quantities of protein drug candidates for in vivo evaluation of efficacy in various disease models. Stable HKB11 clones secreting high levels of a tissue factor (TF; 40-50 pg/c/d) and B-domain deleted recombinant factor VIII (BDDrFVIII; 5-10 microU/c/d) were derived under serum-free conditions. The specific productivity for these two proteins from the HKB11 cells was 10-fold greater than those from CHO cells derived under the similar conditions. In conclusion, we have demonstrated that the HKB11 cell line is well-suited for transient and long-term production of recombinant proteins.     

Genetically stable picornavirus expression vectors with recombinant internal ribosomal entry sites

In many respects, picornaviruses are well suited for their proposed use as immunization vectors. However, their inherent genetic instability hinders application for prophylactic purposes. We demonstrate the improved expression and stability of a heterologous insert through a novel vector design strategy that partially replaces noncoding regulatory sequences with coding sequences for foreign gene products.     

Improved bicistronic mammalian expression vectors using expression augmenting sequence element (EASE)

We have recently reported on the isolation of a 5.7 kb segment of Chinese hamster ovary cell genomic DNA, Expression Augmenting Sequence Element (EASE), which when used in bicistronic expression vectors allows the development of stable Chinese hamster ovary cell pools in a five to seven week time period that express high levels of recombinant protein (6–25 μg 10-6 cells/day depending on the protein). In the present study, we have mapped the activity of the EASE to a 2.1 kb region using colony forming assays and developed bicistronic expression vectors with the smaller EASE or control lambda DNA. The recovery of pools expressing the hematopoietic growth factor, FLT3 Ligand, in methotrexate-containing media took 1 to 4 weeks less when using EASE expression vectors compared with control vectors. The cell pools developed with the EASE and control vectors had similar final protein expression levels. Southern blot analysis suggested the expression cassette from the EASE containing vectors integrated in tandem arrays arranged in either head to head or head to tail fashion. By contrast, control vectors appeared to integrate with multiple interruptions to the expression vector. Thus, the EASE, within a bicistronic expression vector, appeared to facilitate tandem vector integration and reduce the time required to develop cell pools for protein expression. This revised version was published online in August 2006 with corrections to the Cover Date.     

高效可溶性重组蛋白表达载体的构建

本研究构建了两种高效表达可溶性重组蛋白的原核表达载体。一种载体由HisSUMO序列与pET30a(+)载体连接而成(命名为HisSUMO Express),表达的融合蛋白用Ni-NTA纯化,用SUMO蛋白酶I切割后可获得不留任何残基的重组蛋白。SUMO-蛋白酶I价格较贵,为减少表达蛋白的成本,第二种载体即在His-SUMO和目的序列之间加入羟胺切割位点(命名为HisSUMO Economic)。在HisSUMO Economic中表达的融合蛋白用Ni-NTA纯化,羟胺液切割后可获得仅留一个甘氨酸残基的重组蛋白。以在常规表达载体中难以表达的鼠源成纤维细胞生长因子-21(mFGF-21)为例,经葡萄糖消耗实验检测其活性,验证两种表达载体的效果。结果表明mFGF-21在两种载体中均获得了高效表达,融合蛋白占菌体总蛋白的40%以上,Ni-NTA纯化后的融合蛋白分别利用羟胺切割液和SUMO蛋白酶I切割,纯化的mFGF-21成熟蛋白回收量约为54mg/L,回收率约为6%。经两种载体表达后的mFGF-21蛋白均具有生物学活性,可促进脂肪细胞消耗葡萄糖,为进一步研究提供了基础。     

New pUC-derived expression vectors for rapid construction of cDNA libraries

We have constructed a new series of the pUC-derived plasmids with an extended polylinker obtained from M13tg131 [Kieny et al., Gene 26 (1983) 91-99]. These vectors allowed us to design a simplified version of the method of Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] for establishing complete cDNA libraries. Improvements included easy recovery of the inserted cDNA due to the extended polylinkers; use of these vectors for gene expression in Escherichia coli, and amenability to supercoil sequencing with the universal primers of the M13 system [Chen and Seeburg, DNA 4 (1985) 165-170], which speeds up the identification of positive clones. Moreover, there is no need for an additional linker fragment, as was required by the Okayama and Berg [Mol. Cell. Biol. 2 (1982) 161-170] method. We have successfully used this system to obtain cDNA clones coding for the different chains of the large basement membrane proteins type IV collagen and laminin.     

High-level expression of full-length antibodies using trans-complementing expression vectors

Recombinant antibodies are increasingly used as therapeutics for a wide variety of diseases. Generation of cell lines expressing high levels of recombinant antibody typically requires labor-intensive cloning and screening steps. We describe a mammalian expression system for the high-level production of full-length antibody molecules. It has been shown that the dihydrofolate reductase (DHFR) selectable marker can be divided into two fragments that, with the aid of a leucine zipper, can re-associate to form an active molecule. Using bicistronic vectors, we linked the expression of each antibody chain to the expression of a DHFR fragment. Survival in selective media requires expression of both DHFR fragments that, by virtue of these vectors, also selects for the expression of both antibody chains. Initial pools produced 5 microg of Ab/10(6) cells/d (qP = microg/10(6) cells/d). Expression of each antibody chain in conjunction with a portion of DHFR also leads to concurrent amplification of both antibody chains in the presence of methotrexate, a DHFR inhibitor, and results in a two- to fivefold increase in antibody production with basal qPs ranging from 10-25 ug/10(6) cells/d. Shake-flask cultures of amplified pools produced up to 600 mg/L of antibody in 7 days. This system allows for rapid generation of antibodies without cloning and greatly simplifies selection of cell lines for the production of potential antibody therapeutics.     

rDNA介导的酿酒酵母稳定表达载体的构建

将一段含有质粒ColE1复制起始区（ori）和氨苄青霉素抗性基因（bla）的DNA片段插入到酿酒酵母rDNA片段中部的EcoR I或Hpa I位点之间,在pYES2载体的基础上构建了两个rDNA介导的酿酒酵母稳定表达载体pHBM367E或pHBM367H。将黑曲霉糖化酶基因导入载体pHBM367H,获得表达载体pHBM166。为生物安全性的考虑,pHBM166经Hpa I酶切去掉2.2kb的ColE1 ori和bla片段后转化酿酒酵母Y33菌株,获得不含任何抗生素抗性基因的工程菌。随后,对糖化酶基因在酿酒酵母中的表达、稳定性进行了分析,结果显示,rDNA介导的糖化酶基因在酿酒酵母中的表达呈现不同的剂量效应,挑选高表达的菌株传80代之后仍能保持其产糖化酶的稳定性。     

A novel family of retroviral vectors for the rapid production of complex stable cell lines

The production of stable cell lines is an important technique in cell biology, and it is often the rate-limiting step in studies involving the characterization of the function of novel genes or gene mutations. To facilitate this process, a novel family of retroviral vectors, the pE vector family, has been generated. The retroviral sequences in the pE vectors have been taken from the Moloney murine leukemia virus (MMLV) vector pMFG, which has been shown to express cDNA inserts more consistently and at higher levels than earlier generations of MMLV vectors. These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA. The pE vectors have an episomal design to allow long-term production of high-titer virus without the need for subcloning the producer line. Using a strategy of combinatorial infection followed by combinatorial drug selection, we demonstrate that the pE vectors can be used to generate stable, polyclonal cell lines expressing at least three novel cDNAs in less than 2 weeks. The use of these vectors will thus dramatically accelerate the production of complex stable cell lines.     

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene. Using the M. tuberculosis Ag85B as a reporter of gene expression, we demonstrate that the vectors permit the specific detection of recombinant products expressed in the host species M. bovis BCG. BCG over-expressing Ag85B was a potent inducer of Ag85B-specific T cells in immunised mice, indicating that the C-terminal c-myc tag did not alter the characteristics of the recombinant protein. The versatility of the epitope-tagging vectors was demonstrated by the efficient secretion and detection of recombinant products in BCG. The vectors described in this study will facilitate the expression of foreign proteins in mycobacterial host systems.     

New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.     

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.     

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.