Evaluation of Recombinant Proteins ( D. Pranitha )

CONTENTS

Introduction
Gene expression
Protein Expression and Purification
Production of Recombinant Proteins
Applications
Conclusion
References

Introduction

• Proteins are the most abundant organic molecules of the living system. They have significant role in structural and functional organisation of the cell.

• Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins.

• Recombinant DNA technology involves taking genetic material from one source and recombining it in vitro with another source followed by introducing of recombined material into host cell.

• Once a Recombinant DNA is inserted into bacteria, these bacteria will make protein based on this rDNA.This protein is know as Recombinant Protein.

Gene Expression

Protein Expression and Purification

• Isolation of genes.

• Insertion of isolated gene to expression vector.

• Transfer of recombinant vector into host cell through Transformation.

• Identification and isolation of cells containing recombinant vector.

• Growth of cells through fermentation.

• Isolation and purification of protein.

Production of Recombinant protein

• There are basically two methods for producing recombinant
proteins.

• One is the molecular Cloning a laboratory method used to make recombinant DNA.

• The other method is the Polymerase chain reaction used to proceed the replication of any specific DNA sequence selected .

• The basic difference between the two methods is that molecular cloning incorporates the replication of the DNA within a living cell, whereas PCR replicates DNA in the test tube, without living cells.

Cloning process

• Gene of interest is cut out with restriction enzymes (RE)

• Host plasmid (circular chromosome) is cut with same RE

• Gene is inserted into plasmid and ligated with ligase.

• New (engineered) plasmid inserted into bacterium (transform)

Vectors

• Self-replicating DNA molecules used to transfer foreign DNA segments between host cells.

• An ideal vector should be small in size, with single restriction
endonuclease site.

• Three types of vectors

Plasmids

• Bacteria contain extrachromosomal molecules of DNA called plasmids which are circular.

• pBR322 of E.coli is most popular and widely used plasmid vector.

Bacteriophages

• Bacteriophages or simply phages are the viruses that replicate within the bacteria.

• Phages can accept foreign DNA fragments of 10-20 kb length.

Cosmids

• These are specialized plasmids containing DNA sequence namely cos sites.

• Cosmids can carry larger fragments of foreign DNA compared to plasmids .i.e 20-50kb.

Polymerize chain reaction

• A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA polymerase directed DNA synthesis

• PCR copies a DNA molecule without restriction enzymes, vectors, or host cells .

• Faster and easier than conventional cloning.

• First Step in PCR: Denaturation

1. DNA is heated to break the hydrogen bonds between the two
polynucleotide strands.

• Two single-stranded DNA molecules serve as templates.

Second Step in PCR: Annealing

2. Short nucleotide sequences (primers for DNA replication) are mixed with the DNA and bind to complementary regions on
single-stranded DNA .

• Takes place at lower temperature.

• Primers are 20-30 nucleotides long, synthesized in the
laboratory.

Third Step in PCR: DNA Synthesis

3. The enzyme Taq polymerase is added to synthesize a complementary DNA strand.

• Taq is a DNA polymerase from a bacterium found in hot
springs.

• These three steps make up one PCR cycle .

Bir cevap yazın

Başa dön tuşu