Recombinant DNA and Cloning Vector

Learning Objectives

  • Give examples of various recombinant vectors in molecular biology

    • Plasmids

    • Bacteriophages

    • Viruses

    • Artificial Chromosomes

  • Describe in detail the features of plasmids that make them useful for manipulating fragments of DNA and expression of recombinant proteins, with examples

  • Describe the use of inducible promoters in plasmids

  • Describe the relevant differences between prokaryotic and eukaryotic gene expression requirements

  • Describe the use of gene fusions to improve purity of recombinant proteins

Examples of Recombinant Vectors in Molecular Biology

  1. Plasmids:

    • Small, circular DNA molecules replicating independently of chromosomal DNA.

    • Commonly used in bacterial systems for cloning, gene expression, and protein production.

    • Example: pBR322, pUC19, and pET series for protein expression in E. coli.

  2. Bacteriophages:

    • Vectors derived from viruses that infect bacteria.

    • Lambda phage vectors allow large DNA inserts for genomic libraries.

    • Example: Lambda phage-based vectors for cloning DNA fragments up to 25 kb.

  3. Viruses:

    • Lentiviral vectors (e.g., for gene therapy in mammalian cells).

    • Baculovirus vectors (e.g., for protein expression in insect cells).

    • Example: Lentiviral vectors for stable integration of therapeutic genes in human cells.

  4. Artificial Chromosomes:

    • Allow insertion of large DNA fragments for functional studies or library construction.

    • Examples: Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs).

Features of Plasmids in DNA Manipulation and Recombinant Protein Expression

Essential Features:

  1. Replication Origin (Ori):

    • Allows independent replication.

    • Modified to increase copy number (e.g., ColE1 origin for high-copy-number plasmids).

  2. Multiple Cloning Sites (MCS):

    • Contains unique restriction sites for DNA insertion.

  3. Selectable Markers:

    • Confers resistance to antibiotics (e.g., ampicillin, kanamycin) for plasmid selection.

  4. Promoters:

    • Drive gene expression.

    • Constitutive (e.g., CMV for constant expression) or inducible (e.g., lac operon for controlled expression).

  5. Small Size:

    • Typically 4–10 kb for ease of manipulation.

Example:
pET vectors use the T7 promoter to express recombinant proteins in E. coli under control of the T7 RNA polymerase.

Inducible Promoters in Plasmids

Advantages:

  • Prevent toxic protein accumulation by keeping gene expression off until induction.

  • Allow high-level expression on demand.

Example: Lac Operon System:

  • The lac operator is placed near the promoter.

  • Repressed by the lac repressor protein (LacI) in the absence of IPTG.

  • IPTG binds LacI, derepressing the promoter and allowing transcription of the target gene.

Prokaryotic vs. Eukaryotic Gene Expression

Prokaryotic Systems:

  1. Shine-Dalgarno Sequence: Required for ribosome binding.

  2. No Introns: Coding sequence must be intron-free.

  3. No RNA Capping/Polyadenylation: mRNAs lack these modifications.

Eukaryotic Systems:

  1. Kozak Sequence: Facilitates ribosome recognition of the start codon.

  2. Introns and Splicing: Introns can be present and are spliced during mRNA maturation.

  3. RNA Processing: Requires capping, polyadenylation, and proper transcriptional termination.

Example:
A prokaryotic plasmid with a Shine-Dalgarno sequence and bacterial promoter will not function in eukaryotic cells, requiring redesign with eukaryotic promoters (e.g., CMV) and regulatory elements (e.g., polyadenylation signals).

Gene Fusions for Improved Purity of Recombinant Proteins

Purpose:

  • Simplifies purification of target proteins by adding affinity tags or markers.

Types of Fusions:

  1. His-Tag (6xHistidine):

    • Binds to nickel affinity columns.

    • Eluted using imidazole.

  2. GST (Glutathione S-Transferase):

    • Binds to glutathione affinity columns.

    • Cleavage sites allow tag removal post-purification.

  3. GFP (Green Fluorescent Protein):

    • Used to visualize protein localization and trafficking in cells.

Example:
Using a His-tag to purify a recombinant protein:

  • His-tag is fused to the target gene at the N- or C-terminus.

  • The recombinant protein is expressed in E. coli and purified via nickel chromatography.

Applications of Recombinant Vectors

  1. Protein Production:

    • Insulin, interferons, erythropoietin, and monoclonal antibodies.

    • Example: T7 promoter-based plasmids in E. coli for therapeutic proteins.

  2. Gene Therapy:

    • Lentiviral vectors for introducing therapeutic genes in humans.

  3. Basic Research:

    • Studying gene function and regulation using GFP-tagged proteins.

  4. Agriculture:

    • Creating genetically modified crops using Agrobacterium-mediated transformation.

Let me know if you’d like to dive deeper into plasmid design, expression systems, or a specific application!