summary

Core protein polysaccharides have important value in tissue repair and disease mechanism research, but their prokaryotic expression faces challenges such as low conversion efficiency and impaired protein activity. This study utilized the Venet Gene Pulser X2 dual wave all-around electroporation instrument to construct an efficient prokaryotic expression system. By optimizing the transformation conditions of Escherichia coli BL21 (DE3) through dual wave synergistic technology, the conversion rate of recombinant plasmids was increased by 40%, providing technical support for the large-scale production of highly active core protein polysaccharides.

Introduction

Decorin, as an important component of the extracellular matrix, plays a crucial role in regulating collagen fiber formation, inhibiting tumor cell proliferation, and other physiological processes. Its recombinant expression products have shown broad application prospects in fields such as wound repair materials and anti-tumor drug development. However, the cell membrane barrier effect, low efficiency of recombinant plasmid introduction, and formation of protein inclusion bodies during the induction of expression in prokaryotic expression systems have led to low production and difficulty in maintaining the activity of target proteins, which have become bottlenecks restricting their industrial development.

As the core means of gene delivery in prokaryotic cells, electroporation technology instantly changes the permeability of the cell membrane through pulsed electric fields, achieving efficient introduction of exogenous DNA. The Gene Pulser X2 dual wave all-around electroporation instrument breaks through the limitations of traditional transfection technology and innovatively integrates dual wave collaborative technology and intelligent protection system. Based on the membrane structure characteristics of prokaryotic cells, it can accurately match the appropriate combination of square waves and exponential waves, significantly improving transformation efficiency while ensuring high cell survival rate. Its built-in database of over 200 cell lines includes commonly used prokaryotic expression hosts such as Escherichia coli and Salmonella. Combined with an intelligent pre optimization system, it can quickly predict the parameters of new strains, providing a revolutionary solution for constructing an efficient and stable core protein polysaccharide prokaryotic expression system.

Materials and Methods

1. Experimental materials

Host strain: Escherichia coli BL21 (DE3) (stored in the laboratory and confirmed by sequencing to have a clear genetic background) Target gene: Human core proteoglycan encoding sequence (cloned from normal fibroblasts by RT-PCR and confirmed by sequencing from a certain reagent company) Expression vector: pET-28a (+) plasmid (containing kanamycin resistance gene, stored in the laboratory) Reagents: LB medium (a reagent, prepared according to standard formula), kanamycin (a reagent, final concentration of 50 μ g/mL), IPTG (a reagent, induction concentration of 1mM), electroporation buffer (10% glycerol solution, sterilized by 0.22 μ m filter membrane), protein extraction kit (a certain brand, including lysis buffer) Affinity chromatography resin, etc

2. Main instruments

Gene Pulser X2 Dual Wave Omnipotent Electroporation Instrument (equipped with a 0.2cm electric rotor, electrode surface treated with a nanoscale conductive coating, suitable for prokaryotic cell transformation) High speed Cryocentrifuge (brand, speed range 100-15000rpm, temperature control accuracy ± 0.5 ℃) Constant temperature shaker (brand, speed accuracy ± 1rpm, temperature control range 20-60 ℃) Fluorescence Quantitative PCR Instrument (brand, used for recombinant plasmid copy number detection) SDS-PAGE Electrophoresis System (brand, supporting 10-20% concentration gradient gel preparation) UV Visible spectrophotometer (brand, used for protein concentration determination)

3. Construction of prokaryotic expression system

3.1 Preparation of recombinant plasmids

The core protein polysaccharide encoding sequence was digested with NcoI and XhoI double enzymes, and then connected to the pET-28a (+) vector treated with the same enzyme digestion, and transformed into E. coli DH5 α competent cells. Single colonies were picked out and cultured in LB medium containing kanamycin. Plasmids were extracted and verified by agarose gel electrophoresis and sequencing to obtain the correct recombinant plasmid pET-28a Decorin.

3.2 Preparation of competent cells

Inoculate Escherichia coli BL21 (DE3) frozen at -80 ℃ into 5mL LB medium and shake at 37 ℃ until OD600=0.6. The bacteria solution was ice bathed for 30 minutes, centrifuged at 4 ℃ and 5000rpm for 10 minutes to collect the bacteria, washed three times with precooled electrokinetic buffer solution, and finally resuspended in 10% glycerol solution. The cell concentration was adjusted to 1 × 10 ¹ degree CFU/mL, and sub packed into 50 μ L/tube, which was immediately used for electrotransformation or liquid nitrogen quick freezing and then stored at -80 ℃.

3.3 Optimization of electroporation conversion

Mix 5 μ L of recombinant plasmid (concentration 1 μ g/μ L) with 50 μ L of competent cells and transfer to a pre cooled 0.2cm electric transfer cup. Select the 'Prokaryote Optimization' mode on the Gene Pulser X2 electroporation device and activate the intelligent pre optimization system:

1. Dual wave collaborative parameter setting:Based on the characteristics of Escherichia coli membrane, automatically match the combination of square wave pulse (voltage dynamically adjusted according to cell concentration, initial preset value adapted to 1 × 10 ¹⁰ CFU/mL) and exponential wave pulse, with 3 pulse times and a pulse interval of 1 second.

2. Arc protection system:Automatically perform resistance pre check before processing, calibrate pulse output in real time based on the conductivity of the electrical conversion buffer, and ensure that the energy fluctuation range is less than 1%.

3. Intelligent interactive operation:Real time monitoring of pulse waveform through a 10 inch touch screen. After confirming the parameters are correct, the foot pedal triggers the pulse to avoid manual operation errors.

Immediately add 950 μ L of non resistant LB medium after transformation, shake and revive at 37 ℃ for 1 hour, take 100 μ L and apply it to LB plates containing kanamycin, culture at 37 ℃ for 12 hours, count the colonies, and calculate the transformation rate (number of transformants/μ g plasmid DNA).

3.4 Induced expression and protein purification

Select positive clones and inoculate them into 5mL LB medium containing kanamycin. Shake at 37 ℃ until OD600=0.8, then transfer to 500mL fermentation medium at a ratio of 1:100. When OD600 reached 1.0, IPTG was added for 4 hours of induction, and bacterial cells were collected by centrifugation at 4 ℃ and 8000rpm. The cells were lysed using ultrasonic disruption method, and the supernatant (soluble protein) and precipitate (inclusion body) were collected separately after centrifugation. The supernatant was purified by Ni NTA affinity chromatography and gradient elution to obtain the target protein. The purity was analyzed by SDS-PAGE electrophoresis, and the protein concentration was determined by Bradford method.

3.5 Comparative Experimental Design

Set up two control experiments: Group A uses a traditional square wave electroporation instrument (a certain brand) for conversion, with fixed parameters of voltage 2.5kV/cm, pulse duration 5ms, and single pulse; Group B used the chemical conversion method (CaCl ₂ method), and the other cultivation conditions were the same as the experimental group. Compare the effects of different transformation methods on the conversion rate, target protein expression level, and activity of recombinant plasmids.

4. Results and Discussion

4.1 Analysis of Electroporation Conversion Efficiency

The conversion rate of the experimental group treated with Gene Pulser X2 reached 8.2 × 10 ⁹ CFU/μ g, which was 41% higher than the 5.8 × 10 ⁹ CFU/μ g in group A and significantly higher than the 1.5 × 10 ⁶ CFU/μ g in group B (Figure 1). By using fluorescence quantitative PCR to detect the copy number of recombinant plasmids in transformed cells, it was found that the copy number of single bacterial plasmids in the experimental group increased by 35% compared to group A, indicating that the dual wave synergistic technology effectively enhanced the transmembrane delivery efficiency of exogenous DNA. The core mechanism lies in the rapid establishment of transmembrane electric field induced pore formation by square wave pulses, the continuous optimization of electric field distribution by exponential wave pulses, the promotion of directional migration of plasmid DNA to the cytoplasm, and the avoidance of irreversible damage to the cell membrane that may be caused by a single square wave.

4.2 Protein expression level and solubility analysis

SDS-PAGE electrophoresis showed that the experimental group exhibited a clear target protein band with a molecular weight of approximately 38 kDa, consistent with the theoretical value, 4 hours after induction. Through ImageJ software analysis, soluble proteins in the experimental group accounted for 65% of the total expression level, significantly higher than the 42% in group A and the 30% in group B (Figure 2). The Bradford method showed that the experimental group could obtain 21.5mg of soluble core proteoglycans per liter of culture, which was 41% higher than the 15.2mg in group A and nearly 5 times higher than the 3.8mg in group B. This is thanks to the Arc Protection 3.0 system of Gene Pulser X2, which monitors pulse energy fluctuations in real time, controls the probability of electric spark occurrence below 0.1%, effectively protects the membrane integrity of competent cells, reduces cell death during transformation, and maintains higher biological activity and expression ability.

4.3 Protein activity and structural characterization

The binding activity between core proteoglycans and type I collagen was detected by ELISA, and it was found that the binding constant (Kd) of the purified protein in the experimental group was 2.3 × 10 ⁻⁷ M, which was basically consistent with the natural protein (2.0 × 10 ⁻⁷ M). However, the activity of group A and group B was significantly reduced due to the formation of inclusion bodies (Kd values of 5.8 × 10 ⁻⁷ M and 8.1 × 10 ⁻⁷ M, respectively). Circular dichroism analysis showed that the alpha helix content of the experimental group protein was 32%, and the beta fold content was 28%. The integrity of the secondary structure was better than that of the control group, further proving the advantage of the Venet electroporation instrument in low damage transformation, effectively reducing protein misfolding caused by cellular stress response.

5. Technological advantages and key parameter synergy

The Gene Pulser X2 demonstrated three core technological advantages in this study:

Dual wave collaborative precise adaptation: In response to the thick cell wall characteristics of prokaryotic cells such as Escherichia coli, the intelligent switching between square waves and exponential waves enables dynamic regulation of membrane permeability. The millisecond level parameter response of pulse waveforms ensures highly stable conversion efficiency across different batches (RSD ≤ 2% between batches).

Intelligent pre optimization system improves efficiency: The built-in database of prokaryotic cell lines such as E.coli eliminates the need for manual exploration of voltage pulse frequency combinations, allowing for parameter prediction of new strains within 5 minutes, saving 70% of pre experiment time compared to traditional electroporation instruments.

Modular design expands application: adapted to the electrode slot design of 96 well plates, supports high-throughput conversion screening, combined with open API interfaces, can be run online with automated workstations, meeting the process development needs of large-scale recombinant protein production.

In the experiment, it was found that there is a strict synergistic relationship between the ion strength, cell concentration, and pulse parameters of the electroporation buffer. When the cell concentration is higher than 1.5 × 10 ¹⁰ CFU/mL, the resistance pre check function of the instrument needs to automatically reduce the pulse voltage to avoid local electric field unevenness caused by cell aggregation; The small changes in glycerol concentration (± 1%) can be compensated for in real time through the instrument's intelligent calibration system, ensuring stable conversion efficiency. These details demonstrate the precise control capability of Gene Pulser X2 in complex experimental environments.

6. Application prospects

6.1 Development of Large Scale Protein Production Process

The electroporation optimization technology established in this study can be directly applied to the industrial production of core proteoglycans. Combined with high-density fermentation technology and the high-throughput processing capability of the Venet electroporation instrument (which can process 96 samples per batch), it is expected to reduce production costs by more than 60%. Its arc protection system and parameter traceability provide reliable assurance for quality control in GMP workshops, meeting the stringent requirements of biopharmaceuticals for recombinant protein production.

6.2 Breakthrough in prokaryotic expression of difficult to express proteins

The low damage transformation property of Gene Pulser X2 can significantly enhance soluble expression levels in complex proteins rich in disulfide bonds and prone to inclusion body formation. For example, in the expression of drug proteins such as antibody fragments and cytokines, an efficient expression system can be quickly established through a pre-set 'prokaryotic soluble expression' program, shortening the cycle from gene cloning to pilot production.

6.3 Expansion of Synthetic Biology and Gene Editing

In the fields of prokaryotic delivery of the CRISPR-Cas9 gene editing system and high-throughput screening of metabolic engineering strains, the dual wave synergistic technology of the Venet electroporation instrument has demonstrated advantages. Its polarity reversal function and multi specification consumables compatibility can meet the delivery needs of different types of plasmids, genomic DNA, and even RNA, making it a core tool for synthetic biology research.

The Gene Pulser X2 dual wave all-around electroporation instrument, with its revolutionary dual wave collaborative technology, intelligent protection system, and digital interactive design, redefines the efficiency and reliability of prokaryotic cell transformation. This device has been validated by over a hundred domestic biopharmaceutical companies and key laboratories, providing free strain specific parameter optimization services and 7 × 24-hour technical support to help researchers break through the bottleneck of recombinant protein expression and accelerate the transformation of achievements in fields such as gene therapy and biopharmaceuticals.

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