Recombinant Protein Technology

 The use of experimental techniques to construct DNA molecules containing new genes or new
combination of genes is known as genetic engineering or recombinant DNA technology.
 The proteins that are produced as the product of such genetic engineering or recombinant DNA
technology are called recombinant proteins.
There are certain aspects of producing recombinant proteins which helps in advancement of science.
Those are mentioned below:
1. If the wild type of protein is produced in remarkably low amount, often recombinant protein
technology is applied to obtain large quantity of that protein. A strong promoter can be used to
produce / increase the production of protein. Use of multicopy gene/plasmid can be another
alternative of measuring the production. For example, yeast phosphokinase is produced in bacteria
using multicopy plasmid and found that 80% of the total cellular protein is the protein of interest.
Such high concentration of protein allows easier purification. In some cases like structural study,
grams amount of purified protein is required and recombinant proteins allow to uptake such amount.
2. Site directed mutant proteins are used for functional and structural study of protein. Extensive
characterization of a protein is done by site directed mutagenesis.
3. In biotechnological sense, to use a protein in industrial level, certain properties like large amount
production capacity, safety, stability, and cost effectivity must be present. These can be achieved by
recombinant protein technology.
4. Cellular metabolism can be manipulated by the use of recombinant proteins. A strain cam be
developed to produce high amount of a specific protein which has specific cellular metabolic
function.
Basic steps of gene cloning
Gene cloning is the core process of recombinant DNA technology as well as recombinant protein
technology. The basic steps in a gene cloning experiment are as follows:
1. A fragment of DNA; containing the gene to be cloned, is inserted into a circular DNA molecule
called a vector, to produce a chimera or recombinant DNA molecule.
a. At first, the gene/DNA of interest is separated.
b. Suitable vector for both host and the gene of interest is found out.
c. Restriction digestion of both the gene and the vector using suitable restriction
endonuclease.
d. Ligation of the gene with the vector using DNA ligase.

2. The vector (recombinant DNA molecule) acts as a vehicle that transports the gene into a host cell,
(by the means of transformation) which is usually a bacterium. Although other types of living cells
can be used.
1
3. Within the host cell, the vector multiplies producing numerous identical copies not only of itself but
also of the gene that it carries.
4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and
further vector replication takes place.
5. After a large number of cell divisions, a colony or clone of identical host cells is developed. Each
cell in the clone contains one or more copies of recombinant DNA molecules; the gene carried by the
recombinant molecule is now said to be cloned.

Figure: - The basic steps in gene cloning

DNA library
A collection of clones containing all the DNA fragments from a single source is known as DNA library.
DNA library is of two basic types:
1. Genomic library:
DNA libraries that are produced from total DNA extracted from nuclei and contain all the DNA sequenes
of that species, are known as genomic libraries.

2
2. cDNA library:
DNA libraries that are derived from DNA copies of an RNA population, typically from mRNAs p resent
in a particular cell type and thus corresponding to the genes that are active in that type of cell are known
as cDNA libraries.

Construction of genomic libraries

Genomic libraries can be retaining for many years. Traditionally plasmid vectors and phage vectors
were used to clone DNA fragments, but plasmid and phage vectors cannot clone DNA fragments larger
than 20-25 kb. Now-a-days yeast artificial chromosome (YAC) permitting 1000 kb fragment, bacterial
artificial chromosome (BAC) permitting 500 kb fragments, cosmids etc are used as vectors.

3
 Organisms collected from relevant soruce

Propagated in culture

Cells are collected

Phenol-chloroform extraction method

DNA extracted and purified

Partial digestion of chromosomal DNA, only some restriction sites in each DNA are cut; so a set of
overlapping fragments are produced.

Partially digested DNA is fractionated by gel electrophoresis/density gradient centrifugation.

(phosphatase treatment – to inhibit ligation of two fragments)

Fragments of desired size are then joined to vectors (plasmid, phage,etc.)

Transform to bacteria [ a set of bacterial cells are produces containing overlapping fragments; a
few clones contain entire gene of interest, some contain part of it, most have no part of the gene ]

Expression of clones

Colony blot hybridization performed to pick the perfect clone from a

collection of genomic library.