CHIMERIC PROTEINS

Chimeric protein

The chimeric proteins are also called fusion proteins. These are products of chimeric genes. Chimeric proteins are produced naturally as well by gene recombination methods.

 

Natural chimeric proteins

 The chimeric protein produced naturally is the result of cancer such as the bcr abl fusion gene that produces chimeric protein. It causes chronic myelogenous leukemia.

 Other chimeric proteins that are the result of natural processes are formed due to composite mutations such as

 Gene translocations are when a segment of the gene is translocated to another part of the genome. The newly modified gene now produces chimeric protein.

 Duplication is another method of mutation that results in duplication of a segment of a gene in its position that also results in the formation of the chimeric protein.

 Retrotransposons are viral genes that have been incorporated into the genome in evolution. These are produced by reverse transcription and they can translocate themselves to other positions in the genome. This will result in mutation of the gene and protein if produced will be chimeric protein. 

 A chimeric protein structurally has two or three domains that are its functional part. This function part is produced from different genes of different species they will result in the formation of chimeric genes in cells these must be linked with a linker protein. 

The linker protein can be flexible if it consists of poly glycine and rigid if it consists of Proline amino acids. A chimeric protein can be functional or non-functional. The proteins are produced when the functional domains are kept intact but the regulatory part of the gene is transformed from another gene in this way the function of the protein can be upregulated or downregulated regulated.

Chimeric Gene construction

 Non-functional chimeric proteins are usually produced when the two domains of a protein are taken from different species. In this case, they may do not fold in the proper confirmation so this protein will lose its function.

TYPES OF CHIMERIC PROTEINS

 Based on fusion chimeric protein can be N- terminal fusion protein or C-terminal fusion protein. The N- terminal fusion protein has the novel protein attached to the N- terminal of a protein. The C-terminal fusion protein has the novel protein attached to the C- terminal of the protein.

Fusion proteins

 Why do we need to produce chimeric protein

Chimeric proteins are produced for hormones, antibodies, Secretory proteins, enzymes, or structural components of the cells. 

 Chimeric proteins are produced to increase secretion of the proteins, for example in case of antibodies 

Chimeric proteins are produced to increase the solubility of the protein.

 Chimeric proteins are produced to increase the stability of the protein.

 Functional proteins are produced to upregulate or downregulate the function of the protein.

CHIMERIC PROTEINS OF EUKARYOTES

 If chimeric proteins are produced for eukaryotes they must be having some specific components of Eukaryotic gene expression such as Eukaryotic gene has a special structure it has a promoter, inducer, and structural gene.

 The structural genes are separated from each other by introns, that are removed to produce mature mRNA.If the recombinant chimeric gene does not have this capability, it will not function in the Eukaryotic system,

Further in eukaryotes transcription occur in the nucleus so the gene must be located in the nucleus i.e. it must be integrated with the genome in such a way that it can be transcribed. 

It must have the regulatory part that will help its expression in the Eukaryotic system.

 Further, after transcription mRNA is to be translocated outside the nucleus in eukaryotes. So mRNA must-have cap and tail, this helps the nuclear pore complex to recognize mRNA and transfer it to the cytoplasm. 

 The mature mRNA must be capable of being recognized by the ribosome. it must have a specific leader sequence and it must have the initiator codon.

 Further, there must be Codon Optimisation must be done as some codon in bacteria are more common than the other, such as lysin is coded by AAA only 25% and by AAG 75%. translation will be less efficient mRNA has AAA codon more frequent than AAG codon.

 Steps involved in the production of chimeric protein include

 Gene is selected for which a chimeric protein is to be produced for this BLAST studies are done for a protein and compatibility of proteins are established so that the related genes are selected. 

These genes are then used to generate cDNA library .

 From the cDNA library a chimeric gene is produced.  

 This chimeric gene is used for the production of chimeric protein this chimeric protein is produced by inserting the chimeric gene in some Eukaryotic expression vector.

EUKARYOTIC EXPRESSION VECTOR CHIMERIC PROTEIN PRODUCTION

Expression Vector(Ampr -ampicillin resistance gene,ori- origin of replication,G.I. gene of interest,Lac1- lactose metamolism gene)

 In the expression vector, a chimeric gene must have a promoter and inducer which can be CM4, the Pro Asp tag and his tag, and the thyroxine gene for the thyroxine(G.I.) hormone production.

 The promoter can be a constitutional or specific promotor when it is expressed in specific cells constitutional when the protein is expressed in all body cells.

Gene construct for eukaryotic expression

 CM4 is a general inducer

 Pro Asp tag is for cleavage.

 His tag is for purification and thyroxine is for thyroxine protein production. 

 Chimeric protein genes are digested with arl restriction enzyme this enzyme results in the formation of the complementary site in the gene. The complementary site is joined by a linker gene after joining the two genes they are inserted into the expression vector as Baculovirus expression vector.

 This vector is used to insert the chimeric gene in insects so chimeric protein is expressed in insects. Baculovirus expression vector has left border and right border sequences, a terminating sequence, a promoter, and a gene of interest which is inserted at the site of polyhedrin gene.

 This baculovirus vector is then inserted into insect for the expression of chimeric protein.

Baculovirus expression vector( L- left border,R- right border, P- promotor, T- terminator, GoI- gene of interest)

 The expression vector produced from Bculovirus vector is Bacmid vector . It has an antibiotic-resistant chain bacterial gene for expression multiple cloning sites. These vectors are used for bacterial expression as well insect expression.

Precautions taken in chimeric proteins production

Select proper expression vector.

Select proper expression system i.e. E.coli, yeast, insect or mammalian cells.

mRNA should be tagged for its targeting to specific organelle ; mitocondria, ER, golgi, cell membrane.