Improving the soluble expression of proteins that are difficult to express in a prokaryotic expression system through protein engineering and synthetic biology strategies (2023)

Metabolic engineering

Available online 25 May 2023

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Solubility and folding stability are key issues for difficult-to-express proteins (DEPs) constrained by amino acid sequences and superior architecture, and are addressed by precise amino acid distribution and molecular interactions, as well as by expression systems. Therefore, a growing number of tools are available to achieve efficient DEP expression, including directed evolution, solubilization partners, chaperones, and rich expression hosts, among others. In addition, genome editing tools such as transposons and CRISPR Cas9/dCas9 have been developed and expanded for the construction of engineered expression hosts capable of efficient expression of soluble proteins. Taking into account the accumulated knowledge of essential factors in protein solubility and folding stability, this review focuses on advanced technologies and tools for protein engineering, protein quality control systems, and redesign of expression platforms in prokaryotic expression systems, as well as advances in cell-free expression technologies for production of membrane proteins.


Difficultly Expressed Proteins (DEPs) include standards such as toxoids, enzymes and membrane proteins (MPs), enzymes, antibodies, and some proteins that are poorly soluble in prokaryotic expression systems. DEPs have become the most important targets for their therapeutic potential, not only to gain deep insight into functional, structural and biochemical research, but also to develop them as therapeutic proteins, as drug carriers (Hasan et al., 2022) . antibodies (Lloyd et al., 2021), antibacterial agents (Yan et al., 2021) and vaccines (Grund et al., 2021; Marjuki et al., 2019). Currently,Escherichia coliBase-based expression hosts are responsible for a significant portion of DEP production due to their relatively simple and accessible properties (Gupta and Shukla, 2016). However, several problems, including inherently low solubility, low yield, structural instability, and host toxicity, remain highly recursive and difficult to eliminate (Kim et al., 2020). The stability and solubility of proteins in water is determined by a wide range of aspects of cellular microbiology. Central to the water solubility and stability of DEPs are their protein folding homeostasis and quality control (PQC); some misfolded proteins can bypass the proteostasis machinery and remain in a soluble formlive(Nissley et al., 2022.).

To address these obstacles, significant attention has been paid to the positive effects of directed evolution (Wang et al., 2018), soluble factors (Hon et al., 2021) and modified hosts in previous studies. The engineered expression platform can be re-engineered for better performance, although significant engineering strategies already offer sustainable effects on soluble forms of DEP. Consequently, other prokaryotic expression systems, inclBrevibacillus(Yao i sur., 2020.),Bacillus subtilis(Feng i sur., 2017.) ilactic lactococcus(Frelet-Barrand, 2022), have been widely used to produce soluble forms of heterologous proteins. Recently, several reviews have focused on the solubility of DEP in water. For example, Qing et al. (2022) pointed outagaindesign rules for transmembrane proteins, design of stable functional complexes, and computational methodologies for water solubility and structural stability. Liu et al. (2022) summarized protein secretion, modification and transport pathways for efficient protein expression inShepherd's figs. Xiang et al. (2021) reviewed high-density fermentation technologies to improve collagen production. In this review, we focus on summarizing the latest diversification strategies and technologies to improve the production of soluble DEPs. Furthermore, we describe the latest progress and detailed protocol of the CFE technique for PM production.

paragraphs of sections

Protein engineering to improve folding stability

Numerous efforts, exploring methods based on evolutionary and rational design, have been made to engineer the amino acid sequence of DEP to improve folding stability with minimal disruption of function. Charge, polarity, and interaction with surrounding amino acid molecules, as well as pH, temperature, metal ions, cofactors, and surfactants are key factors affecting their apparent stability in water or on a membrane (Kaur et al., 2018; Qing et al. al., 2022

Cell-free expression (CFE) system

Cellular expression of r proteins is sometimes limited by the toxicity of heterologous proteins to the host and their degradation by proteases. The CFE system can be used as an alternative technology to overcome the disability of live cell expression systems. Protein synthesis can be initiated by the transcriptional and translational machinery in cell extracts, with the addition of substrates, DNA templates, and energy substrates necessary for vitro(Libicher e

Conclusions and perspectives

In recent years, prokaryotic expression systems have been shown to yield a wide variety of high-quality r-proteins in a scalable and cost-effective manner. This review presents a multitude of approaches and advances in improving the soluble expression of DEPs in common prokaryotic expression systems. The solubility and stability of proteins are the basis of all life forms for the performance of their intrinsic functions. Understanding the folding mechanism and model allows us to decipher it


This work was financially supportedNational Natural Science Foundation of China(Yes.32171261), oChina's National Key Research and Development Program(Yes.2021YFC2100900), that's onBasic research funds for central universities(Yes.JUSRP22047).


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    What are the strategies of protein engineering? ›

    There are three major approaches of protein engineering research, namely, directed evolution, rational design, and de novo design. Rational design is an effective method of protein engineering when the threedimensional structure and mechanism of the protein is well known.

    Which technique is not used in protein engineering? ›

    Which of the following technique is not used for Protein Engineering? Explanation: DNA fingerprinting is the technique that is not used for protein engineering. DNA shuffling, Error-prone PCR, and rDNA technology are all used in protein engineering.

    What is protein engineering in biotechnology? ›

    Protein engineering is the process by which a researcher modifies a protein sequence through substitution, insertion, or deletion of nucleotides in the encoding gene, with the goal of obtaining a modified protein that is more suitable for a particular application or purpose than the unmodified protein.

    Why are proteins engineered? ›

    Protein engineering is a method in which the protein sequence is deliberately changing to achieve improvements in substrate specificity or increase the stability in a wider range of pH, temperature, or tolerance to a variety of organic and inorganic solvents, or study the specific function of an amino acid [134].

    What techniques and methods are used to determine protein structure? ›

    To determine the three-dimensional structure of a protein at atomic resolution, large proteins have to be crystallized and studied by x-ray diffraction. The structure of small proteins in solution can be determined by nuclear magnetic resonance analysis.

    What is the best method for protein analysis? ›

    In addition, in accordance with the FAO, the amino acid analysis method provides the most accurate measurement of protein content in foods and should be used where possible.

    Which technique is used to determine the expression of a protein? ›

    A technique called mass spectrometry permits scientists to sequence the amino acids in a protein. After a sequence is known, comparing its amino acid sequence with databases allows scientists to discover if there are related proteins whose function is already known.

    What are two methods that can be used to study protein expression? ›

    There are two methods that are commonly used to identify proteins: Edman Degradation and Mass Spectrometry.

    Can you engineer a protein? ›

    Protein engineering is the process by which a researcher modifies a protein sequence through substitution, insertion, or deletion of nucleotides in the encoding gene, with the goal of obtaining a modified protein that is more suitable for a particular application or purpose than the unmodified protein.

    Can biotechnology be used in protein synthesis? ›

    Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative.

    What is the difference between protein engineering and protein design? ›

    In a commonly used definition, the discipline 'Protein Engineering' describes the (more or less) targeted modification of already existing proteins, for instance, to enhance their biophysical or biochemical properties, whereas 'Protein Design' aims at generating artificial proteins displaying novel forms and ...

    What are the challenges of protein engineering? ›

    Challenges in automated protein design include: (i) the enormous search spaces for the optimal sequence and conformation given a defined structure or function, (ii) the development of fast, approximate yet accurate scoring functions that mimic 'true' folding and/or molecular interaction potentials, and (iii) the choice ...

    What are the four properties of a protein that can be changed by protein engineering? ›

    The newly designed protein as a biocatalyst should have modified specific properties that include not only better stability, larger substrate range, higher catalytic efficiency, but also specificity even for the non-natural substrates as well.

    Why is it important for proteins to be built correctly? ›

    Proteins that fold improperly may also impact the health of the cell regardless of the function of the protein. When proteins fail to fold into their functional state, the resulting misfolded proteins can be contorted into shapes that are unfavorable to the crowded cellular environment.

    How are proteins made synthetically? ›

    Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides.

    What are the techniques in protein quantification and analysis? ›

    Protein quantification techniques can include bicinchoninic acid assay (BCA), variations of high-performance liquid-based chromatography (HPLC) and the use of fluorescently labelled or radio-chemically labelled proteins.

    How does genetic engineering allow us to produce a protein? ›

    Therefore, by manipulating DNA, we can potentially modify the structure, function, or activity of proteins and enzymes, which are the final products of gene expression. This concept forms the basis of many genetic engineering techniques such as recombinant protein production and protein engineering.

    Is protein engineering and genetic engineering same? ›

    Generally speaking, protein engineering can be described as the the second genetic engineering that is based on genetic analyses.

    What proteins can be made by genetic engineering? ›

    Engineered Proteins
    • Amino Acids.
    • Eicosanoid Receptor.
    • Cysteine.
    • Protein Engineering.
    • Extracellular Matrix.
    • In Vitro.
    • In Vivo.
    • Energy Transfer.

    What is the process of protein synthesis in prokaryotes? ›

    How does protein synthesis occurs in prokaryotes? In prokaryotes, which lack a nucleus, the processes of both transcription and translation occur in the cytoplasm. The protein biosynthesis begins by the association of a 30s ribosomal subunit and an mRNA at the AUG codon site.

    What is the mechanism of protein synthesis in a prokaryotic cell? ›

    Protein synthesis in prokaryotes is the process that the cell uses to make proteins. It works by taking coded information from the cell DNA and using it to assemble amino acids into proteins. The two main steps of protein synthesis are transcription and translation.

    Why is protein synthesis important for biotechnology? ›

    The purpose of protein synthesis is to make proteins for the cell and for the body. Proteins are important for carrying out chemical reactions, creating structures, acting as signaling molecules and more.

    What are the different types of protein in biotechnology? ›

    There are seven types of proteins: antibodies, contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins, and transport proteins.

    What is the difference between molecular engineering and genetic engineering? ›

    Molecular engineering is a science discipline. It is a culmination of methods and theory advancement that allows us to do certain things and achieve certain goals whereas genetic engineering is results of implementing molecular biology tools.

    What makes a protein difficult to express? ›

    However, some proteins remain difficult to express due to poor growth of the host cell, inclusion body formation, protein inactivity, and low production yield [11].

    What are the factors affecting protein protein interaction? ›

    Protein-protein interaction

    Important affinity factors are the electrical charge of the surface, the hydrophobicity of the protein surface, the chemical functional groups on the pro- teins, the conformational stabilities and the sizes of the proteins.

    What are two factors that can affect protein structure? ›

    Many factors affect the process of protein folding, including conformational and compositional stability, cellular environment including temperature and pH, primary and secondary structure, solvation, hydrogen bonding, salt bridges, hydrophobic effects, van der Waals (vdW) forces, ligand binding, cofactor binding, ion ...

    Which modification approach is an easiest for protein engineering? ›

    Multivalent proteins are relatively easy to produce by post-translational modifications or multiplying the protein-coding DNA sequence.

    Which two factors can lead to a change in shape for a protein and cause its activity to slow down? ›

    Temperature: Raising temperature generally speeds up a reaction, and lowering temperature slows down a reaction. However, extreme high temperatures can cause an enzyme to lose its shape (denature) and stop working. pH: Each enzyme has an optimum pH range. Changing the pH outside of this range will slow enzyme activity.

    Which is the most important structure that determines the functionality of a protein? ›

    The sequence of amino acids determines each protein's unique 3-dimensional structure and its specific function. Amino acids are coded by combinations of three DNA building blocks (nucleotides), determined by the sequence of genes.

    Which protein structure is most important in determining the function of a protein and why? ›

    No two proteins with different amino acid sequences (primary structure) have identical overall structure. The unique amino acid sequence of a protein is reflected in its unique folded structure. This structure, in turn, determines the protein's function.

    What is the most important feature of a protein that causes it to take the correct 3 dimensional structure? ›

    Hydrogen bonds in a protein molecule. Large numbers of hydrogen bonds form between adjacent regions of the folded polypeptide chain and help stabilize its three-dimensional shape.

    What helps in synthesis of proteins? ›

    Nitrogen is the element used for protein synthesis and is an essential constituent of amino acids.

    How can you speed up protein synthesis? ›

    Consuming protein prior to and after the exercise seems to be warranted. Ten grams of essential amino acids or twenty-five grams of a complete protein are sufficient to maximally stimulate protein synthesis. Type, timing and amount of protein are all factors in maximizing muscle mass.

    What substance is used in protein synthesis? ›

    RNA, or ribonucleic acid -- A single-stranded nucleic acid found in the cell nucleus and cytoplasm, which plays a key role in protein synthesis.

    What are the strategies of genetic engineering? ›

    The DNA fragments to be cloned are called foreign DNA or passenger DNA. The DNA fragment of known function is selected and identified. It is isolated from the organism by several in vitro biochemical methods. In addition, the DNA fragments can be constructed chemically by using mRNA of gene machine.

    What are some strategies to prevent protein adsorption? ›

    BSA (bovine serum albumin): One of the easiest ways to prevent protein adsorption is to add BSA to solvents or wash plasticware with a BSA solution. BSA coats plastics and glass very well and largely prevents proteins of interest from adsorbing.

    What are the 3 main tools in genetic engineering? ›

    The basic tools are enzymes, vectors and host organisms. Now we know from the foregoing discussion that in order to generate recombinant DNA molecule, certain basic tools are necessary for the process. The basic tools are enzymes, vectors and host organisms.

    What are the three techniques of genetic engineering? ›

    Commonly used methods include:
    • microinjection of DNA into the nucleus of anchored cells;
    • electroporation, where DNA is introduced through cell membrane pores by pulsed electrical charges;
    • polycationic neutralization of the cell membrane and the DNA to be introduced to improve passive uptake;

    What are the basic techniques used to influence genetic material? ›

    Basic techniques used in genetic material manipulation include extraction, gel electrophoresis, PCR, and blotting methods.

    How do you reduce protein solubility? ›

    Change the pH of the solution.

    Since proteins are least soluble when the pH of the buffer used is equal to the pl of the protein (i.e., the net charge on the protein = 0), changing the pH of the current buffer will charge the net charge of the protein and modulate the interactions that may lead to aggregation.

    What is the most efficient way to absorb protein? ›

    Complex Carbohydrates

    By consuming carbohydrates with your protein, your body releases insulin. Elevated insulin levels help your muscles absorb amino acids, especially during muscle-building exercises. That means eating carbohydrates right before a high-intensity workout yields the best protein-absorbing results.

    How do you increase protein concentration in a solution? ›

    Proteins can be concentrated by precipitation from solution with ammonium sulfate, polyethylene glycol, organic solvent, trichloroacetic acid, potassium chloride/sodium dodecyl sulfate, and three-phase partitioning.


    1. Recombinant protein
    (Shomu's Biology)
    2. Expi293: New Tools for Structural Biology and Difficult to Express Proteins
    3. Strategies for Improving Soluble Protein Production in E. coli
    4. Expression Systems
    (UGA Master of Biomanufacturing and Bioprocessing)
    5. Recombinant Protein Expression in Ecoli
    6. Expressing Insoluble Proteins in a Soluble Form with SoluBL21 Competent E. coli


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