Protein expression and purification experiments

Experimental principle of protein expression and purification

(1) Prokaryotic expression using E. coli expression
The ultimate goal of genetic engineering is to efficiently express foreign genes in a suitable system to produce valuable protein products.

1. Characteristics of prokaryotic gene expression
   (1) Prokaryotes (such as E. coli) have only one RNA polymerase, which recognizes prokaryotic promoters and can catalyze all RNA synthesis.
   (2) The gene expression of prokaryotes is in units of operons. The operon is a combination of several related structural genes and their regulatory regions, and is a coordinated unit of gene expression.
   (3) Since prokaryotes have no nuclear membrane, transcription and translation are coupled, and the two are also continuous. Prokaryotic chromosomal DNA is naked circular DNA. After being transcribed into mRNA, it can be directly combined with ribosome in the cytoplasm and translated into protein. Each ribosome can independently complete the synthesis of one chain, and polyribosomes can simultaneously synthesize multiple peptide chains in one mRNA, greatly improving translation efficiency.
   (4) Prokaryotic genes do not contain introns and there is no post-transcriptional processing.
   (5) The control of gene expression in prokaryotes is mainly at the transcription level. There are two ways to regulate RNA synthesis: initiation control (promoter control) and termination control (attenuator control).
   (6) At the ribosome binding site of E. coli mRNA, there is a translation initiation codon and a sequence complementary to the 3’terminal base of 16S ribosomal RNA, namely the SD sequence, which is not available for eukaryotic genes.

2. The key to the expression of foreign genes in prokaryotes
   (1) The expression vector introduces the foreign gene into the host bacteria, and guides the host bacterial enzyme system to synthesize the foreign protein;
   (2) Foreign genes cannot carry introns;
   (3) Use prokaryotic cell strong promoter and SD sequence to control the expression of foreign genes;
   (4) After the foreign aid gene is connected to the expression vector, the correct open reading frame must be formed;
   (5) Use the control system of the host bacteria to regulate the expression of foreign genes and prevent the expressed foreign gene products from poisoning the host bacteria.

3. Important regulatory elements for the expression of foreign genes in prokaryotic cells
   (1) Promoter
   -35 region: located 35bp upstream of the transcription initiation site, generally composed of 10bp, combined with RNA polymerase delta subunit;
   Region -10 (TATA box): Located 5-10 bp upstream of the transcription start site, generally 6-8 bp, rich in AT, and combined with the core enzyme of RNA polymerase.
   The promoter used in the prokaryotic expression vector must be a prokaryotic promoter. Commonly used strong regulated promoters are: lac (lactose promoter), trp (tryptophan promoter), λPL (λ phage left-facing promoter), Tac (hybrid and promoter of lactose and tryptophan) etc.
   (2) SD sequence
   The translation rate of mRNA in bacteria is strictly dependent on the SD sequence and the distance between the SD sequence and the start codon AUG. For example, when the SD sequence of the lac promoter is 7 nucleotides away from the AUG, IL-2 expression is highest, It is 2581 units, and the expression level drops to less than 5 units when separated by 8 nucleotides. In addition, the binding of certain proteins to the SD sequence also affects the translation of the protein.
   (3) Terminator

4. Types of prokaryotic expression vectors
   (1) Non-fusion type
   An expressed protein that is not fused with any protein or polypeptide of bacteria.
   Advantages: very close to the natural protein in the body;
   Disadvantages: It is easily destroyed by bacterial proteases, and unknown proteins are difficult to purify.
   (2) Fusion type
   One end of the protein is encoded by a prokaryotic DNA sequence or other sequence, and the other end is encoded by the complete sequence of eukaryotic DNA.
   Advantages: The destruction of bacterial proteases should be avoided. Due to the fusion part, it is often easy to separate and purify the expressed product.
   Disadvantages: Due to the presence of a section of bacteria proteins or peptides, it sometimes affects its structure and needs to be removed.

5. Foreign genes for prokaryotic cell expression
   Because prokaryotic cells lack a post-transcriptional processing system for eukaryotic cells, mRNA introns cannot be excised, mature mRNA cannot be formed, and prokaryotic cells also lack a post-translational processing system for eukaryotic cells. Only cDNA can be used instead of genomic DNA, or in vitro synthesis of genes, PCR amplification of genes, etc.
   (2) Purification of ZsGreen protein using HIC resin
   Methylated hydrophobic reaction chromatography (HIC) resin is a simple and effective method for protein separation using hydrophobic groups. In combination with Cl- in the buffer, the β-shell of the negatively charged ZsGreen molecule is repelled. This repulsion turns the molecule inside out, exposing the hydrophobic group. The exposed hydrophobic group binds firmly to the non-polar methyl group of the HIC resin; neutral salt elution keeps ZsGreen in a hydrophobic state; but can elute unbound or weakly bound proteins; low salt buffer makes The hydrophobic group returns to the original position of the ZsGreen molecule, so that the ZsGreen molecule is released from the HIC resin.
   (3) SDS-PAGE analysis
   Polyacrylamide gel electrophoresis is a network structure with a molecular sieve effect. It has two forms. One is a non-denaturing polyacrylamide gel. The protein remains intact during electrophoresis, and the protein is separated according to three factors: protein Size, shape and charge. SDS-PAGE only separates proteins based on the differences in protein molecular weight subunits. This technology was first established by Shapiro in 1967. They found that after adding ionic detergents and strong reducing agents to the sample medium and acrylamide gel, the electrophoretic mobility of protein subunits mainly depends on the size of the subunit molecular weight and the charge factor Can be ignored.
   SDS is an anionic detergent, as a denaturant and a solubilizing agent, it can break the intra- and intra-molecular hydrogen bonds, unfold the molecules, and destroy the secondary and tertiary structures of protein molecules. Strong reducing agents such as mercaptoethanol and dithiothreitol can break the disulfide bonds between cysteine residues. After adding reducing agent and SDS to the sample and gel, the molecule is depolymerized into a polypeptide chain, and the depolymerized amino acid side chain and SDS combine to form a protein-SDS micelle, which has a negative charge that greatly exceeds the original protein of the protein. Quantity, which eliminates the difference in charge and structure between different molecules. SDS-PAGE generally uses a discontinuous buffer system, which can have a higher resolution than a continuous buffer system.
   The role of the concentrated gel is to have a stacking effect. The gel concentration is small and the pore size is large. The thinner sample is added to the concentrated gel and concentrated to a narrow zone after the migration of the large pore gel. When the sample solution and the concentrated gel are selected as Tris-HCl buffer solution, the electrode solution is selected as Tris-glycine. After electrophoresis starts, HCl dissociates into Cl-, and glycine dissociates out a small amount of glycine ion. The protein is negatively charged, so it moves to the positive electrode together, where Cl- is the fastest, glycine ion is the slowest, and the protein is centered. At the beginning of electrophoresis, the Cl-mobility rate is the largest, exceeding the protein, so a low conductivity region is formed behind, and the electric field strength is inversely proportional to the low conductivity region, thus generating a higher electric field strength, causing the protein and glycine ion to move quickly, forming The stable interface allows the protein to gather near the mobile interface and concentrate into an intermediate layer.
   In this experiment, we first extracted the E. coli genome, PCR amplified the cold shock protein CspA promoter, upstream regulation and downstream 5′-UTR region sequences to obtain cspA1, cspA2 and cspA3, and introduced enzymes at both ends Cleavage site; cutting the plasmid pZsGreen1-1 with restriction enzymes to obtain the green fluorescent protein ZsGreen reporter gene; using pET-28a (+) as a backbone, digesting and ligating to construct a cold shock promoter and a reporter gene as ZsGreen The expression vectors pCspA1-ZsGreen, pCspA2-ZsGreen, pCspA3-ZsGreen (Figure 1) and the control normal temperature expression vector pT7-ZsGreen .

Experimental instruments and medicines (1) Preparation of STE (Table 3-1)

 Table 3-1 STE (100mL) formula

Add 5mol/L NaCl 2mL, 1mol/L Tris-HCl (pH8.0) 1mL and 0.5mol/L EDTA (pH8.0) 200μL to 60mL dH2O, mix well, add dH2O to 100mL, 1.034×105Pa high pressure Steam sterilize for 15min and set aside The prepared STE contains 0.1mol/L NaCl, 10mmol/L Tris-HCl and 1mmol/L EDTA.

 (2) Preparation of solution I (Table 3-2)

 Table 3-2 Solution I (100mL) formula

Add 0.9g glucose, 1mol/L Tris-HCl (pH 8.0) 2.5mL and 0.5mol/L EDTA (pH 8.0) 2mL to 60mL dH2O, mix well, add dH2O to 100mL, 1.034×105Pa high pressure steam Bacteria 15min, stored at 4 ℃. The prepared solution I contains 50 mmol/L glucose, 25 mmol/L Tris-HCl and 10 mmol/L EDTA.

 (3) Preparation of solution II (Table 3-3)

 Solution II contains 0.4mol/L NaOH and 1% SDS. This reagent needs to be freshly prepared. After preparation, NaOH and SDS are mixed in the ratio of 1:1.

Table 3-3 Solution II (1mL) formula

(4) Preparation of solution III (Table 3-4)

 Table 3-4 Preparation of Solution III (100mL)

The prepared solution III contains 3mol/L KAc and 5mol/L glacial acetic acid (pH 4.8).

Experimental method of plasmid extraction

The plasmid extraction process is as follows:
 (1) Transfer the positive single colonies on the agar culture plate to 3mL LB liquid medium (containing Amp or Kana), or connect the glycerol bacteria of Escherichia coli to the LB liquid medium at a ratio of 1:30, 37 Incubate vigorously at ℃ overnight;
 (2) Take 500 μL of bacterial cells, mix with 40% glycerol in equal volume, and store at -70℃. The remaining bacterial cells are used for plasmid extraction;
 (3) Remove 1.5-2mL of bacterial solution and transfer it to an Eppendorf tube, centrifuge at 12000rpm, 4℃ for 30s, discard the supernatant, use 1mL STE to suspend the bacterial cell pellet, wash the bacterial cell, and then centrifuge to recover the bacterial cell;
 (4) Repeat rinsing the cells with STE, after centrifugation, remove the supernatant;
 (5) Suspend the bacterial pellet in 100 μL of ice-cold solution I, mix vigorously by shaking;
 (6) Add 200μL of freshly prepared solution II, gently invert the centrifuge tube 5 times to mix the contents, do not shake vigorously, and place on ice for about 3min (according to different strains, it can be shortened appropriately);
 (7) Add 150 μL of ice-cold solution III, invert gently 5 times, mix well, and place on ice for 5 min;
 (8) Centrifuge at 15000rpm, 4℃ for 5min, and transfer the supernatant to another centrifuge tube;
 (9) Add an equal volume of phenol:chloroform (1:1) to the supernatant, mix well, centrifuge at 15000 rpm for 5 min, and transfer the supernatant to another centrifuge tube;
 (10) Add 2 volumes of ice-cold absolute ethanol, leave at room temperature for 2 minutes to precipitate double-stranded DNA;
 (11) Centrifuge at 15000rpm, 4℃ for 5min;
 (12) Discard the supernatant to make the liquid drain as much as possible, and dry and settle in the air;
 (13) Dissolve the nucleic acid pellet with 20 μL of TE buffer containing RNaseA, instantaneously centrifuge, mix well, and store in a refrigerator at -20°C until use.

1. Experimental results (1) Unpurified SDS-PAGE analysis Escherichia coli (E.coli) containing pCspA1-ZsGreen, pCspA2-ZsGreen, pCspA3-ZsGreen was expressed at 16°C; E.coli containing pT7-ZsGreen (E.coli) was expressed at 37°C until OD600=0.6, IPTG was added to induce expression, and those without IPTG were used as controls. The cells were collected after 5 hours of induced expression and analyzed by SDS-PAGE (Figure 5-2).

2. 

3. In Figure 5-2, M is the low molecular weight protein maker. 1-5 are the expressions of pT7-ZsGreen (IPTG-, 37℃), pT7-ZsGreen (IPTG+, 37℃), pCspA1-ZsGreen (16℃), pCspA2-ZsGreen (16℃), pCspA3-ZsGreen (16℃) ZsGreen. The arrow shows ZsGreen, its size is about 27KD. The results of SDS-PAGE showed that the three low-temperature induced expression vectors pCspA1-ZsGreen, pCspA2-ZsGreen, and pCspA3-ZsGreen expressed ZsGreen normally at 16℃, which can be used as the next step to analyze their translation efficiency.

(2) SDS-PAGE analysis after purification
    Escherichia coli (E. coli) containing low-temperature induced expression vectors pCspA1-ZsGreen, pCspA2-ZsGreen, pCspA3-ZsGreen was expressed at 16°C, and the bacterial cells were collected at a time interval of 1h, and the ZsGreen protein was purified using HIC resin for SDS -PAGE analysis (Figure 5-3).

In Figure 5-3, M is the low molecular weight protein maker. A1-7, B8-14, and C15-21 are pCspA1-ZsGreen, pCspA2-ZsGreen, and pCspA3-ZsGreen, respectively, and expressed at 16℃ for 4h. After HIC purification, SDS-PAGE. The arrow shows ZsGreen, its size is about 27KD