Dairy Biotechnology

Module 5. Application of biotechnology in dairying

Lesson 22
CLONING AND EXPRESSION OF GENES IN BACTERIA AND YEAST

22.1 Introduction

Bacteria can serve as factories for production of industrially important native proteins /recombinant proteins. Recombinant DNA technology has been employed to construct/ design strains that over-produce native proteins or foreign proteins. The standard protocols of strain construction involve use of strong promoters, insertion of multiple copies of expression cassettes into the genome and, in some cases, secretion of a foreign protein as a fusion with a native, secreted protein. However, for economical large industrial scale production, further manipulations are required.

22.2 What is a Recombinant Protein?

A protein obtained by introducing recombinant DNA into a host cell causing it to produce the gene product or a protein whose amino-acid sequence is encoded by the cloned gene is called as recombinant protein.

22.3 Basic steps in Recombinant Protein Production

PCR amplification of gene of interest if from prokaryotic source or isolation of RNA/ mRNA and cDNA synthesis if from eukaryotic source

Cloning of amplified PCR product in a PCR cloning vector like pGEM-T or pUA (pDrive)

Transformation into E. coli

Sequencing of the cloned insert

Sub cloning into expression vector

Transformation into expression host e. g. E coli or yeast

Confirmation of identity of recombinant protein using SDS-PAGE, western blot or any other enzyme assay available for a particular gene

Large scale production in fermenters

Downstream processing and purification of recombinant protein

All the above steps are described in the following section:

22.3.1 PCR amplification of gene of interest

22.3.1.1 Procaryotic source

The gene sequence encoding the protein to be expressed is retrieved from the NCBI database available as the public domain. Primers are designed from the flanking region of the gene sequence and used for amplification of the gene of interest from the genomic DNA of the prokaryotic donor.

22.3.1.2 Eucaryotic source

In case of eukaryotic system, RNA is isolated from the donor and cDNA is synthesized using random primers or oligo dT or specific primers as shown in Fig. 22.1. The cDNA is then amplified using the specific primers.

Fig. 22.1 Synthesis of first strand of cDNA from RNA / mRNA

All the above mentioned products are then purified using PCR product purification kits available from several manufacturers e.g. Promega, Qiagen, Invitrogen, Sigma, Banaglore Genei etc.

22.3.2 Cloning of PCR product in a PCR cloning vector like pGEM-T or pDrive

The purified PCR products obtained as above are cloned using PCR cloning vectors e.g. pGEM-T or pUA (TA or UA based cloning).

22.3.3 Transformation into E. coli

The recombinant vector is then transformed into competent cells of E. coli.

22.3.4 Sequencing of the cloned insert

The transformants are picked up and the recombinant plasmid vector carrying the cloned gene as insert is isolated and purified using plasmid purification kits. The nucleotide sequence of the inserted gene is determined and analysed.

22.3.5 Sub cloning into expression vector

Once the sequence of the target or insert DNA matches with the original gene sequence, it can be sub cloned further into an expression vector (for production of protein form the gene of interest.

22.4 What are Expression Vectors?

Expression vectors are the vectors that allow the exogenous / insert / target DNA to be inserted, stored and expressed.

Expression vectors are the basic tools used for the production of proteins of food and pharmaceutical applications such as chymosin, lactoferrin, insulin and streptokinase etc. that are important for food/industrial applications and medical treatments of specific diseases.

22.4.1 Components of an expression vector

An expression vector consists of the following elements important for the production of proteins as shown in Fig. 22.2.

• Origin of Replication (ColE1) – responsible for replication of plasmid DNA.

• Selection marker (ampicillin, kanamycin or chloramphenicol resistance) – antibiotics used as selection markers in order to select recombinant clones / transformants.

• Multiple cloning site (MCS) – comprises of several restriction enzyme sites for cloning the gene of interest

• Transcriptional promoter (T7, T5, T3 or tac) - DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. Strong promoter causes mRNAs to be over expressed at high frequency.

• Intact ORF and ribosomal binding sites (RBS) near start codon are required for translation

• Fusion tags at N terminal or C terminal ends.

• Terminator sequence to terminate transcription.

• Regulatory genes for controlled expression of the target protein.

22.4.2 Strategies for the construction of expression vectors

• Vectors that can synthesize pure proteins exclusively encoded by inserted gene (Transcriptional Fusion)

• Vectors which can allow the synthesis of fusion proteins encoded by the sequence in the vector and those encoded by the inserted gene (Translational fusion)

22.4.2.1 Prokaryotic expression vector

Plasmid derived vectors – A typical plasmid vector has been shown in Fig.22.3.

Fig. 22.3 Genetic Map of pET vector from Novagen

22.4.2.2 Eukaryotic expression vector

Yeast Expression vectors – A vector for expression in methylotrophic yeast Pichia pastoris has been shown in Fig. 22.4.

Fig. 22.4 Genetic Map of pPICZ A, B, C vector from Invitrogen

Other common eukaryotic expression vectors include
Baculo-virus expression vectors
Mammalian expression vectors (SV40, Retrovirus and Adenovirus vectors)

22.5 Transformation into Expression Host e. g. E coli or Yeast

After the construct has been designed, it is transformed into expression host compatible with the vector.

22.5.1 Expression systems

Expression systems are based on the insertion of a gene of interest into a host system (prokaryotic or eukaryotic) for its efficient translation and expression into a protein.

Host expression systems are of the following types:

Procaryotic system – Bacteria like E. coli, B. subtilis, Lactobacillus, Lactococcus etc.
Yeast expression system
Cultured insect cells
Cultured mammalian cells
Transgenic Animals (mammary gland as bioreactors)

22.5.1.1 Procaryotic / Bacterial expression systems

The great demand for production of high amounts of pure protein for pharmaceutical applications and for research made Escherichia coli as one of the most important cell factories for recombinant protein production. Although, it is a well studied model organism showing high productivity, the recombinant protein frequently aggregates and forms the so called inclusion bodies. Inspite that several strategies have been designed to solubilize these inclusion bodies, the recovery of biologically active protein is very low. However, E. coli, the model organism, is the most commonly used expression host for the production of recombinant proteins because of the several advantages.

• Non –pathogenic

• Fast growth (less generation time) approx. 20 min.

• The genome has been well characterized

• Choice of a large number of commercially available vectors

• Can be transformed easily using calcium chloride induced transformation

• The recombinant proteins can be purified using simple techniques

• Can produce recombinant protein at a very high level – gm/L

1. Advantages of prokaryotic expression systems

• Gene expression can be easily controlled by using inducible vectors.

• It is easy to grow the bacteria with high yields.

• The protein can be secreted into the medium.

2. Disadvantages

• Bacteria may recognize the proteins as foreign and destroy them.

• Absence of post translational modifications required for eukaryotic genes expression

• Bacterial environment sometimes may not permit correct protein folding and leads to production of inactive protein

• Eucaryotic proteins when expressed at very high levels form inclusion bodies

• Biological activity and immunogenicity may differ from natural protein.

The most common E. coli host systems are E. coli BL21, E. coli BL2(DE3) and origami using pET series of vectors from Novagen.

22.5.1.2 Eukaryotic expression vectors

• Proper Post translational machinery is present e. g. glycosylation, adenylation

• Disulphide bond formation.

• Proper protein folding.

• Endotoxin free recombinant protein.

22.5.2 Expression in yeasts

Yeasts offer a number of advantages as expression systems for complex mammalian proteins. They combine the ease of manipulation and growth of unicellular organisms to an eukaryotic subcellular organization enabling post translational processing and modification. The two most common hosts used for gene expression in yeasts include Saccharomyces cerevisiae and Pichia pastoris. Some of the examples of commonly used vectors for Saccharomyces cerevisiae are pYES2, pYES2.1/V5-His-TOPO, pYC, pFLAG etc. Recently, Pichia pastoris has been developed into a highly successful system for the production of a variety of proteins. The increasing popularity of this particular expression system can be attributed to several factors which include 1) the simplicity of techniques required for the molecular genetic manipulation of Pichia pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most thoroughly characterised experimental systems in modern biology 2) The ability of Pichia pastoris to produce foreign proteins at high levels either intracellularly or extracellularly 3) The capability of performing many eukaryotic post translational modifications such as glycosylation, disulphide bond formation for correct folding and proteolytic processing and 4) The availability of the expression system as a commercially available kit. Currently, Pichia pastoris expression system has been developed for commercial application by SIBIA, CA with selling rights given to Invitrogen Inc. The most common Pichia expression vectors are pPIC9, pPICZαA, B, C and pGAPZαA, B, C etc. and the hosts are P. pastoris X-33 etc. Many proteins have been expressed in the Pichia Expression System, including enzymes, proteases, protease inhibitors, receptors, single-chain antibodies, and regulatory proteins.

22.6 Confirmation of Recombinant Protein using SDS-Page, Western Blot or any other Enzyme Assay Available for a Particular Gene

Analysis of crude as well as purified product for the presence of target protein is an essential step. Recombinant protein recovered after large scale production and downstream processing also needs to be confirmed once again using these methods.

22.6.1 SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility. The migration of proteins depends on both charge and size. It is basically used for checking purity of the proteins and also determine their molecular weight. Electrophoretic mobility is dependent on molecular weight, higher order protein folding and post translational modifications. A wide range of proteins can be separated by preparing varying concentrations of polyacrylamide gels. Generally, 10% polyacrylamide concentration of gel is sufficient enough to resolve the proteins ranging from 10-150 kDa.The protein mixture to be analyzed is mixed with an anionic detergent such as SDS along with a reducing agent like mercaptoethanol or dithiothreitol (DTT). SDS denatures secondary, tertiary and quaternary structures of proteins and applies a net negative charge to each protein in proportion to its mass. Mercaptoethanol and DTT denature all the disulfide bonds. The protein samples are boiled before loading onto the polyacrylamide gel. Boiling of the protein samples further denatures the protein molecules. SDS then binds strongly along the polypeptide chain and provides a net negative charge to it. Generally, one molecule of SDS binds two amino acid residues. When the electric current is applied, the negatively charged protein migrates from cathode to anode relative to their size. A tracking dye (BPB, Bromo phenol blue) is added to the protein solution to allow the tracking of the progress of the protein solution through the gel during the electrophoretic run. Two different types of apparatuses are shown in Fig. 22.5. Finally, the gels are stained with Coomassie Brilliant Blue R-250 (CBB) or with silver stain.

Fig. 22.5. SDS-PAGE apparatus

22.6.2 Western blot

Western blotting or protein immunoblotting is a technique which is used to identify and locate proteins based on their ability to bind to specific antibodies. It is a widely used technique for confirmation of expression of recombinant protein. Proteins separated onto SDS-PAGE are transferred to Nitrocellulose or PVDF (polyvinylidene difluoride) membrane either by capillary action by stacking or electroblotting. The transferred recombinant or target protein is detected using specific primary antibody and secondary enzyme labeled antibody and substrate using colorimetric, fluorescent, chemiluminescent or radioactive labeling etc.

Some of the other techniques which include application of antibodies to detect proteins are immuno-staining and enzyme-linked immune-sorbent assay (ELISA).

22.6.3 Enzyme-linked immunosorbant assay (ELISA)

ELISA also called enzyme immunoassay is a very common and powerful method extensively used for estimating protein, antigen or antibody in quantities upto even ng/ml to pg/ml in a solution. ELISA is also extensively used in diagnostics. The protein (antigen) detection method is basically the same as in western blot except that it is carried out in 96 well microplate. The sample containing recombinant protein or any other sample is immobilized onto microtitre plate and then detected by the specific antibody.

22.6.4 Other enzyme assays available for a particular gene

The expression of the recombinant protein can also be detected by using enzyme assays if available for that particular protein like β-galactosidase, protease, cellulase, milk clotting activity etc.