03-高效蛋白表达系统

高效蛋白质表达系统1. 原核表达系统 (1)Gram negative (E. coli) (2)Gram positive (B. subtilis) 2. 真核表达系统 (1) Yeast (2) Baculovirus (3) Drosophila (4) Mammalian cells (COS, CHO, BHK etc.) 3. 其它 (1) Cell-free system (2) Protoblast

Protein Expression

Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into amino acid chains, which are ultimately folded into proteins. In its simplest form, a protein expression system involves a template, a mechanism of transcription/translation such as a cell or cell extract, and the raw materials required to build proteins. Protein expression can be done in prokaryotes if the protein doesn’t need to be post-translationally modified. If performing structure-function studies, expressing in a eukaryotic system is important because these cells have the proper cellular machinery to decorate the protein in question with the correct posttranslational modifications. The types of eukaryotic cells typically used in protein expression include yeast, insect, and mammalian.

Choice of the expression systemCell-free Bacteria Yeast Insect Mammalian

Easy of use

Cost of media and Equipment Pos-translational Modifications (Probability of protein function) Time Requirement

Prokaryotic Expression

Protein expression in prokaryotic system

Bacterial expression vectors have some distinct features:

Inducible promoter systems; Protein fusions including fused tags; Easy manipulation;

Some problems of production in E. coli

Inclusion bodies (most common case) Inclusion bodies are formed through the accumulation of folding intermediates rather than from the native or unfolded proteins. It is not possible to predict which proteins will be produced as inclusion bodies. Production of inclusion bodies are not dependent on the origin of proteins, the used promoters, the hydrophobicity of target proteins...

Some E.coli expression host considerations

Yeast Expression

Yeast Expression

Yeast is a eukaryotic organism that can be grown to very high densities, which makes them especially useful for the production of isotope labeled protein for NMR. Useful strains include Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris. The yeast strains have been genetically well characterized and are known to perform many posttranslational modifications. Yeast can grow quickly in defined medium, are easier and less expensive to work with than insect or mammalian cells, and are easily adapted to fermentation. Yeast expression is ideally suited for large-scale production of recombinant eukaryotic proteins. The major advantages of yeast expression: high yield and productivity, high cell densities, and superior expression.

Yeast Expression (Cont.)

Controllable process The growth medium that feeds yeast is completely defined. It consists of a simple, inexpensive formulation. T

he carbon source is fed to the fermentor at a rate designed to achieve maximum cell density while maintaining optimal production of foreign protein. This process minimizes any toxic effects the foreign protein might have on the yeast. Product processing similar to mammalian cells The yeast expression system produces mammalian-like proteins. For example, the expression of Hepatitis B surface antigen (HBsAg) in yeast leads to production of particles that are immunoreactive with anti-HBsAg antibodies. These particles are similar to Dane particles isolated from the sera of human carriers. Stable production strains Expression of foreign genes is achieved by integration of foreign DNA into the chromosomal DNA of host genome. The integrated DNA is stable for many generations; all cells can produce the protein. In contrast, plasmid-based systems require selective pressure on plasmids to maintain the foreign DNA. Cells that lose the plasmid cannot produce the desired foreign protein. Durability The Yeast Expression System requires no special handling. It was developed to withstand the adverse conditions of large scale, continuous fermentors. This feature makes yeast able to survive unexpected disruptions in the fermentation process Lower protein production cost High per-cell expression levels combined with high cell-density growth of yeast translates into greater quantities of recombinant protein per fermentor volume. This reduces production costs by increasing the amount of product per fermentation run. Protein purification is another cost-saving area. The yeast system can secrete protein into the medium, so the broth that enters purification contains a higher concentration of the desired protein. Pure protein is recovered with higher yield and lower cost.

Expression in Yeast

Autonomous replicating vectors -> shuttle vectors

Expression in Saccharomyces cerevisiaeAutonomous replicating systems

Expression in Saccharomyces cerevisiaeIntegrative systems

Probability for integration higher with linear fragments !

Expression in Saccharomyces cerevisiae

Expression in S. cerevisiae Vs. Pichia pastorisProblems with production in S. cerevisiae:

For some proteins production level is low. Hyperglycosylation (more than 100 mannose residues in N-glycosylation). Sometimes secretion is not good -> protein stack in cells (periplasma). S. cerevisiae produces high amount of EtOH -> toxic for the cells -> affects level of production.

Advantages of production in Pichia pastoris:

Highly efficient promoter, tightly regulated (alcohol oxidase -> AOX, induced by MeOH). Produces no EtOH -> very high cell density -> secretion very efficient. Secretes very few proteins -> simplification of purification of secreted proteins.

Insect ExpressionBaculovirus or Drosophila

Baculovirus

Baculovirus are present in invertebrates primarily insect species. They are not infectious for vertebrat

es & plants. Genome is covalently closed circular double stranded of 134 kbp, due to its small it can accommodate large fragments of foreign DNA. They are divided into two groups on the basis of their structure as Nucleopolyhedroviruses (NPV) and Granuloviruses. These NPV are mainly used as expression vectors, i.e. Autographa californica NPV (AcMNPV) isolated from the larva of the alfalfa looper. Baculovirus expression system based upon the ability to propagate AcMNPV in insect cells. Uses many of the protein modification, processing and transport systems present in higher eukaryotic cells. Virus that can be propagated to high titers adapted for growth in suspension cultures obtain large amounts of recombinant protein with relative ease. Baculovirus are noninfectious to vertebrates and their promoters are inactive in mammalian cells.

Advantages of working with Baclo system

High expression levels using the polyhedrin or p10 promoter Supports post-translation modifications BEVS enables simultaneous expression of multiple genes Expressed proteins do not have size limitations Capable of producing cytotoxic proteins

Leukemia in working with BEVS

Baculovirus system works only in invertebrates so the expressed vertebrate proteins are different in post translation modifications with high mannose type glycosylation. It has limited capacity to properly processed inactive precursor proteins due to the absence of pro-protein convertases Limited protein yield due to accumulation of insoluble protein within the cells

Insects & Insect cells

Baculovirus infects lepidopteran (butterflies & moths) insects and insect cell lines. Commonly used cell lines are sf9 & sf21 derived from the pupal ovarian tissue of the fall army worm spodoptera frugiperda and high five derived from the ovarian cells of the cabbage looper.

Types of Insect cell linescells Doubling time Cell appearance Medium Origin Type of culture

Sf 9

72 hrs

Spherical, granular, regular in size, firm attachment to surface

TNM-FH

IPLBSF-21 cell lines of the fall army worm spodoptera frugiperda

Grow well as monolayer and suspension

Sf 21

24 hrs

Spherical, granular, different in size, firm attachment to surface

TNM-FH

IPLBSF-21 cell lines of the fall army worm spodoptera frugiperda Ovarian cells of cabbage looper

Grow well as monolayer and suspension

High-five

18 hrs

Spherical, granular, regular in size, loose attachment to surface

Express five SFM

Grow well as monolayer, also as suspension

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