Background

Overview of Microbial Fermentation for Research-grade Proteins

The roots of microbial fermentation go way back, originally used for making food and drinks. Fast forward to the early 20th century, and this tech started dabbling in protein production. The big leap happened in the 1970s with genetic engineering paving the way for recombinant proteins. Now, critters like E. coli, yeast, and fungi are the main players in churning out these proteins.

Today's microbial fermentation has come a long way with advancements that hike up yield and purity. Think high-density cultures, continuous fermentation, and automation—all making protein production more efficient and economical, helpful for breakthroughs in biopharma and enzyme engineering. Merging history with modern tech, microbial fermentation is now a powerhouse in protein production.

Looking ahead, it's only getting better! Expect more efficiency, precision, and eco-friendliness. Gene editing will boost how microbes produce and express proteins, and synthetic biology is set to craft even better strain platforms. Plus, smarter automated systems will cut down on mistakes and save on labor. With data-driven process tweaks and real-time monitoring, production will get leaner and greener.

Fig. 1. Integrated overview of the global context, substrate utilization, and the sustainable benefits of microbial alternative proteins. (Wankhede, et al., 2024)

Applications of Microbial Fermentation in Research

Microbial fermentation is a big deal in research, especially when it comes to biotech and pharma. By harnessing microorganisms to churn out super pure proteins, scientists can dive into various studies that push forward new drug discoveries, basic science understanding, and biotechnological breakthroughs. Here are some application introductions.

• Enzyme production. In basic research and industrial applications, microbial fermentation is used to produce proteases, amylases, and transglycosylases, etc., which are used as catalysts to promote biochemical reactions and improve experimental efficiency.
• Expression and purification tools. Batch production of labeled proteins, providing convenient protein separation and identification methods, simplifying purification processes, and supporting various biological experiments.
• Protein structure research. Used to produce high-purity tenant proteins, which are further used in crystallography and NMR studies to provide structural information to help scientists understand protein functions and their interactions with other molecules.
• Functional protein design. In biosynthesis and metabolic engineering, the production and application of new molecules are promoted by producing customized enzymes or pathway components.

Creative BioMart Microbe is committed to producing high-purity, high-activity research-grade proteins through microbial fermentation, providing strong support for scientific research and technological development. If you have special protein production or identification requirements, please feel free to contact us for more information.

Service Procedure

Service Details

Strain Development and Optimization

We offer personalized services to develop strains just for you! We handle everything from creating and tweaking target protein genes to building custom expression vectors. By harnessing the latest in molecular biology, we make sure the strains we pick can express the research-grade proteins you need, efficiently and reliably. Our expert team will partner up with you from start to finish, giving you all the support you need—from gene cloning to strain verification—to make sure your project hits the mark.

Intelligent Fermentation Process Control

Our fermentation services feature cutting-edge smart bioreactors that keep a tight rein on crucial factors like temperature, pH, and dissolved oxygen. With real-time data checks and feedback adjustments, we fine-tune the conditions to get the best out of protein expression and ramp up yield and quality.

Efficient Protein Purification and Quality Analysis

We provide fast and dependable protein purification using top-tier methods like affinity chromatography, ion exchange chromatography, and gel filtration chromatography. Our process is all about getting the highest purity and recovery rate for your target protein. Plus, our quality control lab is decked out with high-end gear like SDS-PAGE, Western blot, and mass spectrometry. This ensures our purified proteins are analyzed for quality and activity to meet the demanding standards of research-grade proteins.

Large-scale Production and Customized Services

We have the ability to scale up from laboratory scale to industrial scale, and can flexibly adjust the production scale according to project needs. Our production services include not only fermentation and purification, but also customized solutions such as protein modification, stability testing and long-term stability studies to meet your specific research or commercial needs. Our team will work closely with you to ensure the smooth progress of the production process and provide you with high-quality scientific research-grade protein products.

Comprehensive Microbial Fermentation Services

Our comprehensive microbial fermentation services are designed to provide customers with scalable, robust and commercially viable processes to meet future market needs. We support a range of microbial hosts including bacteria, fungi, etc., and tailor flexible and modular plans for customers, from strain selection, fermentation process optimization to commercialization.

Our Advantages

• Rich strain resources. We've got a bunch of effective expression strains like Escherichia coli, Pichia pastoris, and Bacillus subtilis. We'll pick the best fit for protein expression based on what you need.
• Diverse fermentation capabilities. Our setup supports taking projects from small trials to full-scale production. Our fermentation tanks can go up to thousands of liters, fitting different production scales.
• Professional technical team. Our biotech crew knows their stuff—genetic engineering, protein work, purification, and analysis. They're here to give you all the technical support and solutions you need.
• Flexible customized services. We tailor our services to suit your specific needs, covering everything from protein expression and purification to thorough analysis, all to align with your research goals.

Case Study

Case Study 1: The expression of 5 sequence-optimized proteins in E. coli under different induction conditions.

To optimize expression conditions, BL21 (DE3) and Rosetta (DE3) were used as host strains to express 5 proteins. Each recombinant expression plasmid containing protein was transformed into two types of competent cells, respectively. Then, the parameters of induction expression were also optimized, including inducer concentrations of 0.5 M and 1.0 M, induction temperatures at 18°C, 30°C and 37°C, and corresponding induction timepoints for overnight, 8 hr, 4 hr. Additionally, the target proteins were further processed in lysis buffer at pH 8.5 and pH 4.5, respectively. Eventually, the supernatant and precipitates of target protein from different optimization conditions were collected for SDS-PAGE analysis. The results and summaries were shown as follows.

Fig. 2. SDS-PAGE analysis of protein expression in E. coli. A. lysis buffer (pH 8.5); B. lysis buffer (pH 4.5).

Case Study 2: Small-scale (100 ml culture) purification tests in native conditions for target protein expressed in B. subtilis.

In this project, we used His-Tag and nickel resin for protein purification by affinity chromatography. The purification steps included equilibration and binding with PBS pH 7.5, and washing and elution with imidazole nitrite concentration gradient. The purified samples were quality controlled by SDS-PAGE and quantified by Bradford method. Finally, the yield of SUCP protein was about 37.4 mg/L with a purity of 90%; the yield of PHSL1 protein was about 2.64 mg/L with a purity of 75%. All samples were buffer exchanged after purification, with PBS pH 7.5 as the final buffer.

Fig. 3. Purification tests of target protein.

Case Study 3: Fermentation optimization and purification results of P. pastoris expression system.

This project used the P. pastoris expression system to produce recombinant proteins, using two different culture media and methanol feedback culture after the dissolved oxygen reached the highest level. Purification by HiTrap SP HP affinity chromatography showed that the fermentation effect of Culture medium II was better than that of Culture medium I, and the highest bacterial weight and yield were obtained at 144 hours of culture. Ultimately, the second batch of fermentation obtained the highest yield of 65 mg/L in Culture medium II, indicating that further testing is needed to optimize fermentation conditions such as pH and stirring. Thus, the most suitable fermentation conditions were found to increase the yield of the target protein, and the target protein was successfully purified from the P. pastoris system.

Fig. 4. Target protein purification profile in culture medium.

FAQs

Reference:

1. Wankhede L.; et al. Raw material selection for sustainable fermentation-derived alternative protein production: a review. Syst Microbiol and Biomanuf. 2024.