Recombinant Protein

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A heterologous or recombinant protein is a protein produced by a cell whose genetic material has been modified by genetic recombination. A gene encoding a protein of interest is introduced into the genome of the producing species (bacteria, cultured mammalian cells, transgenic animals, etc.). Recombinant proteins can be purified and used for therapeutic, industrial or research purposes.

Composition of the transgene

The vector is a means of transporting DNA. The donor organism expresses the protein of interest. The mRNA encoding this protein is isolated from this organism. MRNA allows the synthesis of the cDNA that will be used for cloning. The RT-PCR technique is often used to obtain the cDNA encoding the gene of interest. Following reverse transcription of the total mRNAs into ssDNA (single stranded) using poly dT primers specific for poly A ends of the mRNAs, the cDNA corresponding to the mRNA encoding the protein of interest is amplified by PCR using specific primers to which restriction sites are added which will then be used for cloning.

Cloning of the gene of interest

Recombinant DNA
The PCR amplified cDNA is cloned into a vector. The cDNA of the gene of interest are digested with different restriction enzymes using a ligase.
The recombinant plasmid is then introduced into a bacterium, by transformation, in order to amplify the vector which will then be purified. Before carrying out the transgenesis, the vector is linearized and the bacterial replication signals as well as the resistance genes present on the plasmid are removed using restriction enzymes.

Transgenesis

It is a technique of integrating an exogenous gene into the genome of a host organism. The purpose of this manipulation is to allow the host to produce a protein of interest not produced by a given species or to produce it in larger quantities (used in research activities)

Choice of the host

It will be based on the desired use of the recombinant protein as well as the cellular mechanisms necessary for the production of a functional protein: Bacteria, Yeasts, Mushrooms, Mammalian cells, Plants, Animals proteins in the diet, requires a large structure for breeding.

Introduction of the gene of interest into the host genome

For bacteria and yeasts, the transgene will either be introduced into the cell on a plasmid. In plants, the integration of recombinant DNA is done through a “DNA gun”, which projects micro-beads of gold or tungsten coated with DNA or by the use of bacteria capable of transferring DNA.
For animals, injection of a solution containing recombinant DNA in linear form is performed in the male pronucleus of an egg cell prior to the karyogamy step.

Recombinant Protein

Composition of the transgene

Cloning of the gene of interest

Transgenesis

Choice of the host

Introduction of the gene of interest into the host genome

Tommy Ounas

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