Molecular Biology. Practical Guide - Velikov V.A. 2013

Isolation of Recombinant Protein
Induced Expression of Cloned Genes

Genes are cloned either with their own regulatory elements (promoters, terminators, etc.) in cloning vectors, or the structural part of the Gene is engineered using Introduction/32.html">Genetic Engineering techniques to fit under a regulated promoter in expression vectors. In this case, Gene Expression is relatively easy to control. Normally, it is typically repressed. Production of the recombinant protein begins only after a specific inducer is added to the nutrient medium for cultivating the producer Cells. Very often, expression vectors utilize the LacZ promoter of the E. coli ß-galactosidase gene (see App. 6). Protein expression from this promoter is induced by lactose or a non-hydrolyzable lactose analog, isopropyl-ß-D-thiogalactopyranoside (IPTG). The addition of glucose to the medium represses METABOLISM/35.html">Protein Biosynthesis.

Certain vector systems that utilize highly processive bacteriophage RNA polymerases instead of the host Cell machinery for Transcription of the cloned gene are also induced using IPTG. For example, in the E. coli strain BL21(DE3), the inducible gene for phage T7 RNA polymerase (recombinant, with a LacZ-type promoter) is located on the host cell chromosome, whereas the target gene under the control of the phage T7 promoter resides on a pET-series plasmid (NovagenETM, App. 1, 2). Such systems completely lack "basal" or Background expression, because transcription and coupled Translation of the cloned gene are possible only after the induced Synthesis of the phage RNA polymerase. Cloning the gene into pET30a, pET30b, and pET30c Plasmids ensures the correct reading frame for the DNA insert, as these plasmids feature an insertion of 0, 1, or 2 NUCLEOTIDES downstream of the phage T7 promoter.

Workshops 9.1–9.3 provide protocols for inducing recombinant Protein Synthesis in E. coli cells using the LacZ promoter, isolating the protein from the bacterial periplasm, and purifying it via dialysis.

Class="center">Workshop 9.1. Induced Expression of cloned Genes

To obtain the target protein, producer clone cells are propagated by inoculating them into a fresh nutrient medium. The medium must contain a selective antibiotic; otherwise, the Bacteria may lose the recombinant plasmid. Production of the recombinant protein begins only after the addition of the inducer. As the bacterial culture grows, the total amount of the target protein increases due to active transcription-translation of the cloned gene and an increase in The Cell population. It reaches saturation in the late logarithmic growth phase of the culture.

A protocol is described for inducing the synthesis of single-chain Antibodies with a specific affinity using superproducer clone cells. scFv antibodies are recombinant Proteins consisting of covalently linked variable domains of immunoglobulin light and heavy chains (scFv — single-chain variable fragments). A superproducer clone is an E. coli JS5 strain clone containing the recombinant phagemid pHEN1 with the gene for a miniantibody specific to the virulence protein VirE2 of the soil bacterium Agrobacterium tumefaciens (Velikov et al., Molecular Genetics, Microbiology and Virology, 2006, No. 1). Recombinant scFvs with the desired Specificity are obtained using Antibody Phage Display technology.

The described methodology can be applied to any other recombinant construct containing the LacZ gene promoter.

Equipment and Materials

E. coli strain JS5 containing the phagemid pHEN1 with the scFv miniantibody gene, centrifuge, shaker.

Solutions

- 2YT medium (Topic 1).

- Ampicillin. 100 mg/mL solution in Water.

- 40% Glucose.

- 1M IPTG. 154 mg/mL solution in water.

- 50 mM NaCl.

Procedure

1. Inoculate E. coli strain JS5 (pHEN1::scFv) cells containing the phagemid with the cloned miniantibody gene into 10 mL of 2YT medium. The medium must contain 100 µg/mL of the antibiotic ampicillin and 1% glucose. To do this, add 10 µL of the ampicillin stock solution and 250 µL of 40% glucose to the culture tube. Grow overnight with aeration at 37°C.

2. Dilute the overnight culture 100-fold with the same medium (transfer 10 mL into a flask containing 1 L of medium) and grow at 25°C until an optical density of 0.5 at 600 nm is reached.

3. Pellet the cells by centrifugation at 4000 rpm for 10 min.

4. Resuspend the cells in 50 mL of 50 mM NaCl solution to wash out residual glucose.

5. Pellet the cells again by centrifugation at 4000 rpm for 10 min.

6. Add the inducer to the washed cells. To do this, resuspend the cell pellet first in a small volume and then transfer it to 1 L of 2YT medium containing 100 µg/mL ampicillin and 1 mM IPTG. To achieve the required final concentrations, add 1000 µL of the antibiotic stock solution and 1000 µL of the IPTG inducer stock solution to the 1-liter flask.

7. Incubate the cells for 3 hours at 25°C on a shaker. Lowering the Temperature compared to 37°C improves recombinant protein accumulation and prevents culture autolysis during overexpression.

8. Stop the growth of the bacterial culture by placing the flask in a refrigerator at +4°C, after which proceed to isolate the recombinant miniantibodies.