Protein Expression in Animal Cells: 43 (Methods in Cell Biology)

Read the latest chapters of Methods in Cell Biology at leondumoulin.nl, Elsevier's leading Volume 43, Pages ii-xvi, () Chapter 2 Expression of Exogenous Proteins in Mammalian Cells with the Semliki Forest Virus Vector.
Table of contents

SearchWorks Catalog

Find it at other libraries via WorldCat Limited preview. Contributor Roth, Michael G. American Society for Cell Biology. Bibliography Includes bibliographical references and index. Compans and Paul C. Naim and Michael G. Weisz and Carolyn E. Lee and Robert W. Willnow and Joachim Herz.

Cell Culture Training Video

Johnston and De-chu Tang. Publisher's Summary Critically acclaimed for more than 25 years, the Methods in Cell Biology series provides an indispensable tool for the researcher. Each volume is carefully edited by experts to contain state-of-the-art reviews and step-by-step protocols.

Techniques are described completely so that methods are made accessible to users. Nielsen Book Data Protein Expression in Animal Cells presents information essential to researchers and students investigating cellular function. This exceptional laboratory guide covers the selection and use of expression systems that are optimal for specific exogenous proteins. Current studies often require protein expression in particular cell types, in amounts suitable for specific detection methods, and under conditions that do not otherwise perturb the specific cellular mechanisms under investigation.

Plasmids are circular DNA molecules that lead an independent existence in the bacterial cells. They are naturally occurring, extra chromosomal DNA fragments that are stably inherited from one generation to another generation in an extra chromosomal state. Adenovirus is medium sized, non-enveloped icosahedral virus. It is composed of a nucleocapsid and a double stranded linear DNA genome that can be used as a cloning vector.

The extensive knowledge and data collected over the years on adenovirus transcription regulation favored the engineering of adenovirus vectors modified for heterologous expression. This makes the virus replication incompetent and this function must then be provided in trans by the host cell. An expression cassette was set in place of the deleted E1 region. In the cassette, the recombinant gene was placed under control of an additional or ectopic major late promoter or under control of an exogenous promoter, such as cytomegalovirus. The fact that the virus can be propagated in suspension cell cultures is of considerable advantage for large scale work.

Reports were made regarding the recombinant adeno associated viral vectors also. The genome of vaccinia virus is made up of double stranded DNA of nearly , bp and replicates in the cytoplasm of the host cell. The vaccinia system has been efficiently used at very large scale L to produce many different kinds of proteins, such as HIV-1 rgp by Pasteur-Merieux 29 and human pro-thrombin by Immuno AG. The reason for making retrovirus as vector lies in the fact that most retroviruses do not kill the host, but produce progeny virons over an indefinite period of time.

Retroviral vectors can therefore be applied to make stably transformed cell lines. The next reason is that viral gene expression is driven by strong promoters, which can be subverted to control the expression of transgenes. The other reason is that some retroviruses, such as amphotropic strains of murine leukaemia virus MLV , have a broad host range, allowing the transduction of many cell types. It has been reported that exogenous gene expression system based on the retroviral vector is an alternative method for the generation of stable and high-expressing mammalian cell lines.

Baculovirusespossess rod-shaped capsids with large double stranded DNA genomes. They productively infect arthropods, particularly insects.


  • Part I The Use of Naturally Occurring and Recombinant Viruses.
  • The Red Dun Filly;
  • Rivers of Life.

The former is used for protein expression in insect cell lines, particularly those derived from Spodopterafrugiperda e. The latter infects the silkworm for the production of recombinant protein. Construction and application of a recombinant baculovirus containing a bicistronic expression cassette that can be used for stable protein expression in mammalian cells have been reported.

The choice of the vector depends on the method used for the introduction of the foreign gene into the mammalian cells and on the control elements utilized for the efficient mRNA expression and protein synthesis. There are two general methods for the introduction of foreign DNA into mammalian cells. One is mediated by virus infection and the other by direct transfer of DNA into the cells employing chemical liposomes, calcium phosphate, DEAE-de-xtran and polybrene and physical electroporation and microinjection methods.

A number of methods used to transform cell has been also reported by this author also. The first approved biologic from a mammalian bioprocess platform was tissue plasminogen activator tPA , produced in by Genentech Inc. Today, production of biologics in mammalian cells dominates: Mammalian cells are currently the main hosts for commercial production of therapeutic proteins, including monoclonal antibody mAbs. However, they are not the most suitable cells for large-scale processes of production, since they lose the production ability over time. Actually, if the aim is large-scale production, the most currently used cells for this application are CHO cells, which have assumed an increasing importance in laboratory.

Stanford Libraries

Guidelines to cell engineering for mAbs production were also reported. High levels well above the levels seen from parental hybridomas of chimeric antibodies and recombinant antibody fragments have been achieved by using low copy number cell lines. Urokinase is a serine protease that activates plasminogen into plasmin, which in turn degrades fibrin clots.

Hence urokinase finds its value as an important anti-thromboembolic drug. The need for urokinase production has increased significantly in recent years, and current production levels have not kept the same pace. Mammalian cells provide the post-translational modifications; therefore mammalian cell lines are nowadays preferred for production of recombinant urokinase.

CHO cells are suitable hosts for recombinant urokinase production because they grow exceptionally well inside the bioreactor. Follicle-stimulating hormone FSH is produced by the gonadotropic cells of the anterior pituitary. FSH play its role for follicular growth and development in females. It is essential for males for the pubertal initiation and maintenance of quantitatively normal adult spermatogenesis.

Protein expression in animal cells in SearchWorks catalog

Besides their therapeutic application in the treatment of infertility, recombinant gonadotrophins have been used for studying structure and functions of glycoprotein hormones. They are free from contaminating hormones, and their structure and function can be studied extensively because of their availability in large excess. Novel mammalian cell lines expressing reporter genes for the detection of environmental chemicals activating endogenous aryl hydrocarbon receptors or estrogen receptors were reported. Stable and high-level production of recombinant Factor IX in human hepatic cell line was reported.

As it is known that mammalian expression system provides protein which is functional due to glycosylation. In other words protein obtained through gene expression in mammalian cells always remains biological active. In spite of various benefits the mammalian expression has its limitation. As author has good expierence in working in this system. According to this author this system is highly expensive. The complicated technology, and potential contamination with animal viruses of mammalian cell expression have been bottlenecks for its use in large-scale industrial production, this system is often utilized to express many heterologous proteins including viral structural protein and bioactive peptide for specific functional analysis.

Improvement of mammalian expression can be achieved by proper designing of vector. It includes using strong promoter, proper signal peptide, selected introns and product gene codon optimization. Expression systems for recombinant proteins using mammalian cells are able to introduce proper protein folding, post-translational modifications, which are often essential for full biological activity. The main conclusion is that the author has summarized the latest advancements in the field of mammalian expression system which has its importance in animal biotechnology.

The author explained about the cell lines, vectors and the transfection methodology used in mammalian expression system which are highly important for those researchers who are starting their career in animal biotechnology.

Introduction

In last two decades, mammalian cell protein expression has become the dominant recombinant protein production system for clinical applications, producing more than half of the biopharmaceutical products in the market and several hundreds of candidates in clinical development. Significant progress in developing and engineering new cell lines, introducing novel genetic mechanisms in expression, gene silencing, and gene targeting have been achieved. An optimal expression system can be selected only if the productivity, bioactivity, purpose, and physicochemical characteristics of the interest protein are taken into consideration, together with the cost, convenience and safety of the system itself.

It is the present need to discover more mammalian cell lines and vector system and technologies to express the gene to get more efficient proteins having high biological activities. As this author is first and corresponding author so there is no competing interests. Chinese hamster ovary; DHFR: Hhuman embryonic kidney; PEI: Green fluorescent protein; SV Simian viruses 40; CMV: Autographacalifornica multiple nuclear polyhedrosis virus; BmNPV: National Center for Biotechnology Information , U.

Journal List Adv Pharm Bull v. Published online Aug This article has been cited by other articles in PMC. Abstract The production of proteins in appropriate quantity and quality is an essential requirement of the present time. Introduction Gene is defined as a discrete unit of genetic information which is required for the production of a polypeptide.

Table 1 Factors for selecting an expression system. No Factors effecting gene expression 1 Facilities of the laboratory and local expertise.

Protein Expression in Animal Cells

Open in a separate window. Table 2 Key features for most of transient expression systems. No Important features of transient expression systems 1 Extremely short time-frame for the generation of product days 2 Applicability to a wide range of host cell lines 3 Intrinsic genetic stability and consistency due to extremely short time-frame between generation of vector and product recovered. Requirements for mammalian expression system The expression level of a gene largely depends upon how efficiently it is transcribed. Cell lines In the previous decade, protein therapeutics produced from mammalian cells have changed the landscape of human healthcare.

Table 3 Mammalian cell lines. Plasmid based expression vectors Expression vector is a vector that allows the transcription and translation of a foreign gene inserted into it. Adenovirus vectors Adenovirus is medium sized, non-enveloped icosahedral virus. Vaccinia vectors The genome of vaccinia virus is made up of double stranded DNA of nearly , bp and replicates in the cytoplasm of the host cell.


  • Moody Forest.
  • Signature Golf?
  • The Cobra in the Barn: Great Stories of Automotive Archaeology.
  • .
  • .

Baculovirus as vectorso Baculovirusespossess rod-shaped capsids with large double stranded DNA genomes. Process for transfer of gene The choice of the vector depends on the method used for the introduction of the foreign gene into the mammalian cells and on the control elements utilized for the efficient mRNA expression and protein synthesis.

Application of mammalian expression system The first approved biologic from a mammalian bioprocess platform was tissue plasminogen activator tPA , produced in by Genentech Inc. Production of monoclonal antibody Mammalian cells are currently the main hosts for commercial production of therapeutic proteins, including monoclonal antibody mAbs. Production of urokinase Urokinase is a serine protease that activates plasminogen into plasmin, which in turn degrades fibrin clots. Production of follicle stimulating hormonee Follicle-stimulating hormone FSH is produced by the gonadotropic cells of the anterior pituitary.

Other applications of mammalian expression system Novel mammalian cell lines expressing reporter genes for the detection of environmental chemicals activating endogenous aryl hydrocarbon receptors or estrogen receptors were reported. Limitation of mammalian expression system As it is known that mammalian expression system provides protein which is functional due to glycosylation. Improvement of mammalian expression system Improvement of mammalian expression can be achieved by proper designing of vector.

Concluding remarks Expression systems for recombinant proteins using mammalian cells are able to introduce proper protein folding, post-translational modifications, which are often essential for full biological activity. Conflict of Interest As this author is first and corresponding author so there is no competing interests. Gene Transfer Technologies and their Applications: Roles in Human Diseases.

Asian J Exp Biol Sci. Gene transfer technologies in plants: Recent Res Sci Technol. Gene transfer technologies leading to transgenic animals. Recent advances in the production of proteins in insect and mammalian cells for structural biology. Why proteins in mammalian cells? Advancing mammalian cell culture engineering using genome-scale technologies. Protein reference mapping of dihydrofolatereductase-deficient CHO DG44 cell lines using 2-dimensional electrophoresis.

Gene Expression in Mammalian Cells and its Applications

Wurm FM, Hacker D. Optimization of 25 kDa linear polyethylenimine for efficient gene delivery. Baculovirus-mediated gene transfer into mammalian cells. BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies. Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells. CHO cells in biotechnology for production of recombinant proteins: The effects of CD59 gene as a target gene on breast cancer cells. Lentiviral vector-mediated PAX6 overexpression promotes growth and inhibits apoptosis of human retinoblastoma cells.