Recombinant Protein

Recombinant Protein

Recombinant DNA technology is one way of studying the functions and interactions of proteins. This is done by isolating a target DNA sequence and then transferring it to a cloning vector that has the ability to self-reproduce. The recombinant DNA is transferred to RNA, which in turn produces the recombinant protein.
Recombinant DNA technology allows for the production of wild type and modified human and mammalian proteins at bulk quantities. Recombinant proteins are made from cloned DNA sequences which usually encode an enzyme or protein with known function. So recombinant proteins are a new combination of genes that forms DNA.
Recombinant proteins are made through genetic engineering, also called gene splicing or recombinant DNA technology. By putting human, animal or plant genes into the genetic material of bacteria, mammalian, insect or yeast cells, these microorganisms can be used as factories or producers to make proteins for medical, academic and research uses.
A vector is simply a tool for manipulating DNA and can be viewed as a “transport vehicle” for the production of proteins from specific DNA sequences cloned into them. Purification and expression of a protein can sometimes be quite complicated & time-consuming, therefore an additional tag is used in addition to the specific DNA sequence which will facilitate the purification & expresion of the recombinant protein, for example, 6 × His Tag, Fc Tag.
Recombinant Protein is a protein whose DNA has been created artificially. DNA from 2 or more sources which is incorporated into a single recombinant molecule. The DNA is first treated with restriction endonuclease enzyme which the ends of the cut have an overhanging piece of single-stranded DNA. These are called “sticky ends” because they are able to base pair with any DNA molecule containing the complementary sticky end. DNA ligase covalently links the two strands into 1 recombinant DNA molecule.
Recombinant DNA must be replicated many times to provide material for analysis & sequencing. Producing many identical copies of the same recombinant DNA molecule is called cloning. Cloning is done in vitro, by a process called the polymerase chain reaction (PCR). Cloning in vivo can be done in unicellular microbes such as E.coli, unicellular eukaryotes like yeast and in mammalian cells grown in tissue culture.
Recombinant DNA molecule must be taken up by the cell in a form in which it can be replicated and expressed. This is achieved by incorporating the DNA in a vector. A number of viruses (both bacterial and of mammalian cells) can serve as vectors.
Recombinant DNA is also sometimes referred to as chimera. When combining two or more different strands of DNA, there are 3 different methods by which Recombinant DNA is made. 1. Transformation, 2. Phage-Transfection 3.Yeast, Plant & Mammalian Transformation. When using the method of transformation one needs to select a piece of DNA to be inserted into a vector, cut a piece of DNA with a restriction enzyme and ligate the DNA by inserting into the vector with DNA Ligase. The insert contains a selectable marker which allows for identification of recombinant molecules. Antibiotic markers are used in order to cause death for a host cell which does not contain the vector when exposed to the certain antibiotics.
Trasnformation is the insertion of the vector into the host cell. The host cells are prepared to take up the foreign DNA. Selectable markers are used for antibiotic resistance, color changes, or any other characteristic which can distinguish transformed hosts from untransformed hosts. Yeast, Plant & Mammalian Transformations are done by micro-injecting the DNA into the nucleus of the cell being transformed. Phage-Transfection process, is equivalent to transformation except for the fact that phage lambda or M13 is used instead of bacteria. These phages produce plaques which contain recombinant proteins which can be easily distinguished from the non-recombinant proteins by various selection methods.
Significant amounts of recombinant protein are produced by the host only when expression genes are added. The Protein’s expression depends on the genes which surround the DNA of interest, this collection of genes acts as signals which provide instructions for the transcription and translation of the DNA of interest by the cell. These signals include the promoter, ribosome binding site, and terminator. The recombinant DNA is inserted into expression vectors which contain the promoter, ribosome binding site, and terminator.
In prokaryotic systems, the promoter, ribosome binding site, and terminator have to be from the same host since the bacteria is unlikely to understand the signals of human promoters and terminators. The designated gene must not contain human introns since the bacteria does not recognize them and this results in premature termination, and the recombinant protein may not be processed correctly, be folded correctly, or may even be degraded.
The peptide sequence can be added as an extension at the N-terminal. Researchers can select the specific purification system which they would like to use. The unique vectors available contain several features needed for the production of bulk quantities of the target protein. The peptide sequence is usually placed in the vector so that it is designed to be a point of attack for a specific protease. Thus, after the recombinant protein is expressed and extracted from bacteria, specific peptide extension can be used to purify the protein and subsequently removed from the target protein to generate a nearly natural sequence on the final product.
For example, 6 or more consistent Histidine residues act as a metal binding site for recombinant protein purification and expression. The 6 × His sequence is called a His-Tag sequence which can be placed on the N-terminal of a target protein by using vectors from various commercial molecular biology companies. The His-Tag contains a cleavage site for a specific protease. His-Tag recombinant proteins are purified by Metal Chelate Affinity Chromatography. Then the purified His-Tag protein is treated with the specific protease to cleave off the His-Tag or not if the tag doesn’t affect the active site of the protein.
Proteins have metal binding sites which can be used for the purification of recombinant proteins and natural proteins. This type of purification is rather simple when using a gel bead which is covalently modified so that it displays a chelator group for binding a heavy metal ion like Zn2+ or Ni2+. This purification method is quite identical to affinity chromatography when purifying metal-binding class of proteins.
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List of notable recombinant proteins

The following is a list of notable proteins that are generated from recombinant DNA, using biomolecular engineering, focusing on those that are used in human and veterinary medicine. In many cases, recombinant human proteins have replaced the original animal-derived version used in medicine. The prefix “rh” for “human recombinant” appears less and less in the literature.
Many recombinant proteins are used in the research laboratory. These include both commercially available proteins (for example most of the enzymes used in the molecular biology laboratory), and those that are generated in the course specific research projects.
Human recombinant proteins that largely replaced animal or harvested from human types
  • Human growth hormone (r-HGH): Humatrope from Lilly and Serostim from Serono replaced cadaver harvested human growth hormone
  • Biosynthetic human insulin (BHI): Humulin from Lilly and Novolin from Novo Nordisk among others; largely replaced bovine and porcine insulin for human therapy. Some prefer to continue using the animal-sourced preparations, as there is some evidence that synthetic insulin varieties are more likely to induce hypoglycemia unawareness. Remaining manufacturers of highly purified animal-sourced insulin include the U.K.’s Wockhardt Ltd. (headquartered in India), Argentina’s Laboratorios Beta S.A., and China’s Wanbang Biopharma Co.
  • Follicle-stimulating hormone (FSH): as a recombinant gonadotropin preparation replaced Serono’s Pergonal which was previously isolated from post-menopausal female urine
  • Factor VIII: Kogenate from Bayer replaced blood harvested factor VIII
Human recombinant proteins with recombination as only source
Animal recombinant proteins
  • Bovine somatotropin (bST)
  • Porcine somatotropin (pST)
  • Bovine Chymosin
Viral recombinant proteins
  • Envelope protein of the hepatitis B virus marketed as Engerix-B by SmithKline Beecham
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News: Myasthenia gravis therapies

Myasthenia gravis therapies: an interview with Professor Daniel Drachman

http://www.news-medical.net/news/20121115/Myasthenia-gravis-therapies-an-interview-with-Professor-Daniel-Drachman.aspx

Please could you give a brief introduction to myasthenia gravis?
Myasthenia gravis (MG) is an autoimmune disease that produces weakness and fatiguability of muscles. It affects between 1 and 7 people per 10,000, according to the best statistics.

Typically, it may begin with double vision and droopy eyelids. Weakness then often progresses to involve the face, the arms and legs, and the muscles concerned with breathing and swallowing. The name “myasthenia gravis” means serious (grave) muscle weakness.

Before the development of current treatments of the immune system, about two thirds of MG patients did very poorly or died. With present treatment, nearly all MG patients can be brought back to a nearly normal or completely normal life, although there may be problems with the side effects of the treatments.

What is known about the causes of myasthenia gravis?
MG is due to a mistaken response of the immune system that attacks the acetylcholine receptors of muscles. Every muscle movement requires the release of the transmitter acetylcholine (ACh) from motor nerves, which then crosses a very narrow gap to reach the ACh receptors (AChRs), and triggers contraction of muscles. Failure of transmission causes weakness or paralysis.

In MG, there is an attack by the immune system’s antibodies on the AChRs, which are decreased in number. This produces weakness, fatigue, and may cause paralysis and death.

The actual cause of the mistake by the immune system is not known, but there is evidence that a combination of a genetic susceptibility plus some environmental trigger leads to the autoimmune attack. The thymus gland is probably involved in the origin of MG.

What therapies are currently available for myasthenia gravis?
There are now a number of treatments for MG. The most immediate treatment is a drug (pyridostigmine, Mestinon) that blocks the enzyme acetylcholinesterase that normally hydrolyzes ACh after the event of neurotransmission. This drug allows ACh to persist longer, and to interact repeatedly with the reduced number of AChRs. It has a temporary beneficial effect, and is considered a sort of “band-aid” for MG.

Agents that suppress the immune system as a whole are used to treat the underlying autoimmune problem in MG. These include adrenal corticosteroids (Prednisone), and other general immunosuppressive agents, such as azathioprine, tacrolimus and cyclosporine. These agents suppress the whole immune system; properly used they are quite effective. However, they may have adverse side effects, including increased susceptibility to infection, and rarely malignancies, as well as other side effects.

How did your research into developing a gene-based therapy to stop the rodent equivalent of the myasthenia gravis originate? What did your research involve?
The goal of my research is to develop a treatment that will eliminate the specific abnormality that is responsible for the autoimmune problem without otherwise affecting the immune system.

To accomplish this, it is necessary to target those cells that are specifically involved in the autoimmune response. I set out to target the T lymphocytes which provide help for production of antibodies to AChR. So the first aspect of our research involved genetically engineering antigen presenting cells (the most efficient of which are Dendritic cells) so that they would target the T cells that specifically interact with AChRs.

A special issue involved in this is that each individual (or for that matter each mouse) with MG has its own unique set of T cells specific for different aspects of the antigen AChR (different epitopes). To deal with that complication, we would use dendritic cells from the same individual, which of course are capable of targeting the entire repertoire of that individual’s AChR-specific T cells. The targeting gene complex consists of a gene for the most important part of the AChR linked to a complex that directs it to the endosomal processing compartment, so that it is presented on the surface of the dendritic cell.

Once we had perfected the targeting method, we chose a “warhead” to destroy the T cells, and for this purpose we supplied our dendritic cells with the gene for Fas Ligand (FasL). FasL interacts with “Fas”, a protein on the surface of activated T cells, causing death by apoptosis.

The next step was to test the engineered dendritic cells in vitro. We found that they did indeed target and kill AChR-specific T cells, but not T cells that were specific for another unrelated antigen. Having been able to demonstrate the specificity of these “guided missile” dendritic cells, we then tested their ability to kill AChR-specific T cells in living mice.

We immunized the mice with two antigens – AChR and an unrelated antigen (KLH). We injected the mice with the “guided missile” engineered dendritic cells, and evaluated the T cells from the mice, and the production of antibodies in the mice. AChR-specific T cells from the treated mice were markedly reduced, whereas T cells specific for KLH were not reduced at all. Similarly, the antibodies against AChR were highly significantly lower in the Guided Missile treated mice than in those not treated with Guided Missile dendritic cells, whereas the antibody response to KLH was not affected by the AChR-specific Guided Missiles.

There are several special features to these results:

It is important to be able to use the individual’s own dendritic cells, so as to target the entire repertoire of that individual’s autoreactive T cells.
The targeting complex and the “warhead” must both be expressed by all of the dendritic cells. If only the targeting mechanism were present, the dendritic cells would stimulate – rather than eliminating – the T cells. On the other hand, if only the “warhead” were present without the targeting complex, the dendritic cells could damage other T cells, and damage other tissues, such as liver or lungs.
What impact do you think your research will have on Myasthenia gravis therapies and do you think your research will have any impact on therapies for other autoimmune diseases?
Our experiments have demonstrated a “proof of principle” i.e. they showed that the “Guided Missile” strategy of specific immunotherapy can be carried out in vivo to treat the autoimmune response in a model of MG.

Actually, a similar approach could be used for any autoimmune disease for which the antigen is known. I am hopeful that this, or a similar strategy will be translated for treatment of human MG and other autoimmune diseases. Of course, each part of the process must be developed according to “Best Clinical Practices” standards, which will require considerable development.

Do you have any plans for further research into this area?
We are currently studying the genetics of MG by carrying out a Genome Wide Association Study (GWAS). We have collected DNA from 1100 MG patients and some 3,000 non-affected control individuals of similar ethnic background.

In collaboration with Dr. Bryan Traynor at the NIH, we are comparing single nucleotide polymorphisms (SNPs) from MG patients with those from controls. So far we have found 3 SNPs that are highly significantly different in our MG population, and many more that are promising. These are being sequenced, and appear to involve strongly relevant genes.

Where can readers find more information?
The original artricle was published in J. Neuroimmunology 2012 251:25 – 32.

Further information on MG can be found in:

News: Topotarget grants exclusive license to Oncology Venture regarding APO010

To NASDAQ OMX Copenhagen A/S
Investor News No. 07-12 / Copenhagen, November 13, 2012

As Topotarget continues to focus its operations on the company’s lead candidate belinostat, the company today announced that it has entered into an exclusive license agreement with Oncology Venture ApS granting Oncology Venture global rights to the further clinical development of APO010.

APO010 is an anti-cancer drug comprising multimerised Fas ligand/FasL protein, which utilizes Topotarget’s proprietary Mega ligand technology (a non-core component of Topotarget’s IP assets). APO010 targets Fas receptors on the surface of cancer cells and induces cell death via apoptosis. APO010 has shown activity in otherwise drug-resistant cells.

Under the agreement, Oncology Venture will license all rights specific to APO010 which are covered by Topotarget’s issued and pending patents in Europe, the US, Canada, Japan, Australia, South Korea, and other territories. The agreement also grants Oncology Venture the right to sub-license.

Anders Vadsholt, CEO of Topotarget, states: “Topotarget’s focus is on the development of our lead drug candidate belinostat and the license agreement for APO010 with Oncology Venture is in line with this strategy”.

Professor Peter Buhl Jensen, CEO of Oncology Venture, said: “I’m happy to get the opportunity to acquire and develop APO010 for the treatment of oncology patients”.

No details of the terms of the agreement were disclosed.

Today’s news does not change Topotarget’s full-year financial guidance for 2012.
Background information

About Topotarget
Topotarget (NASDAQ OMX: TOPO) is an international biopharmaceutical company headquartered in Copenhagen, Denmark, dedicated to clinical development and registration of oncology products. In collaboration with Spectrum Pharmaceuticals, Inc., Topotarget focuses on the development of its lead drug candidate, belinostat, which has shown positive results in the treatment of hematological malignancies and solid tumors, obtained by both mono- and combination therapy. For more information, please refer to http://www.topotarget.com.

Topotarget Safe Harbor Statement
This announcement may contain forward-looking statements, including statements about Topotarget A/S’ expectations to the progression of Topotarget A/S’ clinical pipeline and with respect to cash burn guidance. Such statements are subject to risks and uncertainties of which many are outside the control of Topotarget A/S, and which could cause actual results to differ materially from those described. Topotarget A/S disclaims any intention or obligation to update or revise any forward-looking statements, whether as a result of new information, future events, or otherwise, unless required by Danish law.

Gene Expression Informatics

Summary

There are many methodologies for performing gene expression profiling on transcripts, and through their use scientists have been generating vast amounts of experimental data. Turning the raw experimental data into meaningful biological observation requires a number of processing steps; to remove noise, to identify the “true” expression value, normalize the data, compare it to reference data, and to extract patterns, or obtain insight into the underlying biology of the samples being measured. In this chapter we give a brief overview of how the raw data is processed, provide details on several data-mining methods, and discuss the future direction of expression informatics.

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Small Amplified RNA-SAGE

Summary

Serial analysis of gene expression (SAGE) is a powerful genome-wide analytic tool to determine expression profiles. Since its description in 1995 by Victor Velculescu et al., SAGE has been widely used. Recently, the efficiency of the method has been emphasized as a means to identify novel transcripts or genes that are difficult to identify by conventional methods. SAGE is based on the principle that a 10-base pair (bp) cDNA fragment contains sufficient information to unambiguously identify a transcript, provided it is isolated from a defined position within this transcript. Concatenation of these sequence tags allows serial analysis of transcripts by sequencing multiple tags within a single clone. Extraction of sequence data by computer programs provides a list of sequence tags that reflect both qualitatively and quantitatively the gene expression profile. Several modifications to the initial protocol allowed to start from 1 μg total RNA (or 10^5 cells). In order to reduce the amount of input RNA, protocols including extra polymerase chain reaction (PCR) steps were designed. Linear amplification of the mRNA targets might have advantage over PCR by minimizing biases introduced by the amplification step; therefore we devised a SAGE protocol in which a loop of linear amplification of RNA has been included. Our approach, named “small amplified RNA-SAGE” (SAR-SAGE) included a T7 RNA polymerase promoter within an adapter derived from the standard SAGE linker. This allowed transcription of cDNA segments, extending from the last NlaIII site of transcripts to the polyA tail; these small amplified RNAs then serve as template in a classical (micro)SAGE procedure. As the cDNAs are immobilized on oligo(dT) magnetic beads, several rounds of transcription can be performed in succession with the same cDNA preparation, with the potential to increase further the yield in a linear way. Except for the transcription step itself, the present procedure does not introduce any extra enzymatic reaction in the classical SAGE protocol, it is expected to keep the representation biases associated with amplification as low as possible.

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Suppression Subtractive Hybridization

Summary

Suppression subtractive hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. In fact, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. The SSH method is based on a suppression PCR effect and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNA or genomic DNA fragments, and simplifies analysis of the subtracted library. In our hands, the SSH technique has enriched over 1000-fold for rare sequences in a single round of subtractive hybridization.

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Amplified Differential Gene Expression Microarray

Summary

Amplified Differential Gene Expression (ADGE) and DNA microarray provides a new concept that the ratios of differentially expressed genes are magnified prior to detecting them. The ratio magnification is achieved with the integration of DNA reassociation and polymerase chain reaction (PCR) amplification and ensured with the design of the adapters and primers. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. The combination of ADGE and DNA microarray provides a mutual complement of their strengths: the magnification of ratios of differential gene expression improves the detection sensitivity; the PCR amplification and efficient labeling enhance the signal intensity and reduce the requirement for large amounts of starting material; and the high throughput for DNA microarray is maintained.

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