Recombinant Protein Articles

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Production of Recombinant Proteins – Challenges and Solutions (PDF)
Abstract: Efficient strategies for the production of recombinant proteins are gaining increasing importance,as more applications that require high amounts of high-quality proteins reach the market. Higher production efficiencies and, consequently, lower costs of the final product are needed for obtaining a commercially viable process. In this chapter, common problems in recombinant protein production are reviewed and strategies for their solution are discussed. Such strategies include molecular biology techniques, as well as manipulation of the culture environment.

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications
Abstract: Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology.

So you Need a Protein – A Guide to the Production of Recombinant Proteins
Abstract: The field of biotechnology owes a great deal to the ability to produce recombinant proteins, which can be made
in far greater abundance than many native proteins, and are more easily quality controlled. There is a great need for individual
proteins to be produced for research purposes. This review is aimed at researchers who are not experienced at protein
expression, but find that they have a need to produce a recombinant protein. We detail the major expression systems
that will be commonly used in the laboratory situation- bacterial, yeast and insect cell culture. The application of each,
and the relative advantages/disadvantages are discussed.

History of Recombinant Protein

Herb BoyerWhat is the History of Recombinant protein? The method of recombinant DNA was initially planned by a graduate student, Peter Lobban, along with a biochemist, A. Dale Kaiser at the Stanford University.

During then years,1972–74, the method was then acknowledged by Stanley Norman Cohen, an American geneticist Chang, Herbert Boyer, a addressee of the 1990 National Medal of Science. In 1973, they published their predictions in journal “Enzymatic end-to-end joining of DNA molecules” which explained the methods to separate and intensify genes or DNA segments and introduce them into an additional cell with accuracy.

In 1977, an advance in the field of recombinant DNA technology took place when Herbert Boyer created the biosynthetic “human” insulin, a group of biosynthetic human insulin products.

 

Recombinant Protein Definition

Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products. The formation of recombinant protein is carried out in specialized vehicles known as vectors. Recombinant technology is the process involved in the formation of recombinant protein.
— recombinant protein definition from http://www.ehow.com/about_5407160_recombinant-protein-definition.html

Recombinant Protein is a protein encoded by a gene — recombinant DNA — that has been cloned in a system that supports expression of the gene and translation of messenger RNA (see expression system). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. Proteins coexpressed in bacteria will not possess post-translational modifications, e.g. phosphorylation or glycosylation; eukaryotic expression systems are needed for this.
— recombinant protein definition from http://www.answers.com/topic/recombinant-protein

Recombinant DNA (rDNA) molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure; they differ only in the sequence of nucleotides within that identical overall structure. Consequently, when DNA from a foreign source is linked to host sequences that can drive DNA replication and then introduced into a host organism, the foreign DNA is replicated along with the host DNA.
Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein will not necessarily be produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence.
— recombinant protein definition from https://en.wikipedia.org/wiki/Recombinant_protein

Protein Expression System

Protein expression system is specifically designed for the production of a gene product of choice. This is normally a protein although may also be RNA, such as tRNA or a ribozyme. An protein expression system consists of a gene, normally encoded by DNA, and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein using the reagents provided. Protein expression system is therefore often artificial in some manner. Expression systems are, however, a fundamentally natural process. Protein expression refers to the way in which proteins are synthesized, modified and regulated in living organisms. In protein expression system, Bacterial expression system, mammalian expression system and yeast expression system are widely used.

– protein expression system introduction from https://en.wikipedia.org/wiki/Gene_expression#Expression_system

The category of the expression system

E.coli protein expression system:

The bacterial protein expression system is the easiest, quickest and cheapest method. There are many commercial and non-commercial expression vectors available with different N- and C-terminal tags and many different strains which are optimized for special applications.

Yeast protein expression system

Yeast is an eukaryotic protein expression system and has some advantages and disadvantages over E. coli. One of the major advantages is that yeast cultures can be grown to very high densities, which makes them especially useful for the production of isotope labeled protein for NMR. The two most used yeast strains are Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris.

Mammalian protein expression system

Most labs use HEK (human embryonal kidney) or CHO (Chinese hamster ovary) cell lines for preparative expression of more complex proteins which also need proper post-translational modifications. Both cell lines can be used for both transient and stable cell line expression which is more time consuming due to the generation of stable cell lines but offers higher productivity and less variation if long-term production of a target protein is required. While these cells have usually a high capacity of producing secreted protein (up to 10s or 100s of mg per Liter, often several grams per Liter in cell lines for commercial proteins), their expression levels for intracellular proteins is usually much lower.

– protein expression system category from https://www.embl.de/pepcore/pepcore_services/cloning/choice_expression_systems/

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Bacterial Expression System

During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial expression systems remain most attractive due to low cost, high productivity, and rapid use. Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins.

– bacterial expression system introduction from:

http://www.sciencedirect.com/science/article/pii/S0734975011001662

Escherichia coli

E.coli is one of the most widely used bacterial expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E.coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting transcription.

Corynebacterium

Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine, components of human food, animal feed, and pharmaceutical products.
Unlike gram-negative bacteria expression systems, the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans.

Pseudomonas fluorescens

The non-pathogenic and gram-negative bacteria expression systems, Pseudomonas fluorescens, is used for high level production of recombinant proteins; commonly for the development bio-therapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapid and successfully produce high titers of active, soluble protein.

– bacterial expression system category from:

https://en.wikipedia.org/wiki/Protein_production_(biotechnology)#Bacterial_systems