Chapter 16, Problem 36P

Summary Introduction

a.

To draw:

The recombinant plasmid that indicates the order of the four components and their arrangement in relation to the plasmid vector.

Introduction:

Erythropoietin, which is a human protein stimulates the production of red blood cells. The idea is to make a bacterium that produces this protein, and this recombinant bacterium can be used for the treatment of anemia patients.

Summary Introduction

b.

To determine:

The element that encodes the ribosome binding site for the mRNA making the fusion protein.

Introduction:

The recombinant plasmid was made to contain coding sequence for human erythropoietin, the regulatory sequence of the lac operon, a sequence encoding E.coli maltose-binding protein, and a sequence encoding a series of five amino acids. These sequences were transformed in the recombinant plasmid to induce the expression of a tagged fusion protein N MBP-DDDDK-erythropoietin C.

Summary Introduction

c.

To determine:

The elements out of given four that should be placed in the same reading frame with respect to each other.

Introduction:

The four elements present in the recombinant DNA molecule include coding sequence for erythropoietin, a sequence encoding the maltose-binding protein in E.coli and a series of five amino acids, and the regulatory sequence for lac operon.

Summary Introduction

d.

To determine:

Whether the erythropoietin coding sequences can be obtained from a human genomic DNA or from a human cDNA clone.

Introduction:

The creation of cDNA clones occurs in three major steps known as the synthesis, cloning, and validation. The cDNA clones contain open reading frames and untranslated regions.

Summary Introduction

e.

To determine:

The compound that could be used for inducing expression of the fusion protein and giving a reason about adding the compound to the medium before seeding with E.coli cells or after reaching the population of cells to high density.

Introduction:

The fusion protein that has to be made in the recombinant E.coli producing erythropoietin protein is N MBP-DDDDK-erythropoietin C.

Summary Introduction

f.

To determine:

The process by which fusion protein can be purified away from the other E.coli proteins.

Introduction:

The cells that express the fusion protein also contain many other E.coli proteins. It is considered to be important to purify the drugs away from the contaminants. It is known that MBP tightly binds to the sugar maltose and this maltose can be attached to an insoluble resin.

Summary Introduction

g.

To determine:

The way by which enterokinase can be used to separate the erythropoietin away from the rest of the fusion protein and then perform the purification of the desired pharmaceutical.

Introduction:

It is been studied that the human erythropoietin should not be attached to any other amino acid sequence. So, a protein known as enterokinase is used to cleave the proteins just C-terminal to DDDDK.