Expression toolkits can significantly boost the chances of successful recombinant protein expression. At Laurus Bio, we’ve crafted specialized toolkits for E. coli, P. pastoris, and H. polymorpha, ensuring high-level protein production. We are also exploring the possibility of establishing expression systems using filamentous fungi. Our toolkit’s effectiveness is showcased in our extensive protein product portfolio https://laurus.bio/our-products/

DNA functions as the blueprint of life, encoding information through chemical entities known as bases. Diversity in the coding and non-coding regions of DNA arise from the varied sequences, permutations, and combinations of its bases¹. The complexity of information escalates as this code is translated into proteins, the cellular workhorses. In contrast to stable DNA sequences that encompass information for protein production, proteins themselves exhibit variable stabilities and demand specific conditions for optimal activity. The universality of the genetic code empowers researchers to express proteins from higher organisms in heterologous systems, including microbes. In 1982, the FDA granted approval for the use of insulin, the first recombinant protein, in the treatment of type II diabetes². After its approval, recombinant insulin supplanted its natural origin (the pancreas of pigs), marking a pivotal moment in the continuous growth of recombinant protein production in industrial biotechnology, therapeutics, and diagnostics. Leveraging recombinant DNA technology for smart protein production³ opens a broader opportunity to potentially substitute animal-derived proteins in a sustainable and environmentally friendly manner.

Despite many years of advancements in the recombinant DNA technology sector, a comprehensive solution for expressing various native proteins in non-natural heterologous systems remains elusive. Hence, it becomes essential to investigate multiple expression systems and associated components to increase the probability of achieving high-level expression of functional proteins (Fig.1).

Fig. 1: Screening and selection of strains expressing recombinant proteins. The components of the protein expression toolkit include different strains (of a heterologous organism), transcription elements, accessory factors, including signal sequences for localization of proteins.

Bacteria and yeast stand out as the most widely utilized heterologous systems for recombinant expression of proteins, with end applications in the therapeutic and food industries.

Escherichia coli

Many researchers prefer utilizing strains of E. coli as the primary choice for expressing recombinant proteins, primarily due to the lower capital investment, shorter lead time, and high yields in comparison to other options. Recombinant proteins can be expressed in the periplasm or cytoplasm (soluble cytoplasmic fraction or inclusion bodies) of E. coli⁴. Co-expression of recombinant proteins in the cytoplasm with a diverse array of chaperones and disulfide bond modulators can aid in the folding of the expressed protein into its native conformation, consequently enhancing soluble expression levels⁵. Functional proteins can be refolded from inclusion bodies by employing high throughput screening methodologies aiding in the identification of optimal refolding conditions⁶.
We present here a case study of a protein of interest, which was found predominantly in the insoluble fraction when expressed in E. coli. Co-expression with chaperones leads to an increased level of soluble expression of the protein of interest under the tested conditions (Fig. 2).

Fig. 2: Co-expression of a protein of interest with chaperones in the cytoplasm of E. coli. M – Molecular weight marker; UI- Uninduced whole cell lysate; W – Induced whole cell lysate; P- Inclusion body fraction; S- Cytoplasmic fraction. Co-expression with a combination of chaperones lead to an increase in the soluble expression of the protein of interest, with chaperone 1&3 giving the best result followed by chaperone 2&3.

At Laurus Bio, growth factors required for growth, maintenance, and differentiation of stem cells, have been successfully produced in E. coli. One such example is IGF1-LR3, which is available in powder as well as in liquid form (Fig. 3). A pipeline of proteins belonging to different functional classes have been produced or is being evaluated in E. coli.

Fig. 3: IGF1-LR3 (Insulin like growth factor 1 – Long repeat 3) expressed in E. coli at Laurus Bio is available in liquid and powder form. For more details, please visit https://laurus.bio/our-products/

Yeast

Komagataella phaffi, more commonly recognized by its former name, Pichia pastoris, is a methylotrophic yeast extensively employed for the heterologous production of numerous enzymes and other proteins. The strain’s designation as GRAS (Generally Recognized as Safe) enhances its appeal as a host for producing proteins applicable in various fields. These include cell culture (e.g., trypsin, fibroblast growth factor basic, human albumin), therapeutics (e.g., insulin, hepatitis B vaccine), animal feed additives (e.g., phytase), and food (e.g., pepsin A, egg white protein), among others⁷. By constructing and assembling expression vectors that incorporate diverse signal sequences, promoters, and terminators, it becomes possible to screen multiple expression conditions, thereby enhancing the likelihood of achieving high-level expression of recombinant proteins. Co-expression with accessory proteins which modulate the folding and redox status of the protein can further enhance the expression levels by preventing misfolding of proteins.

We present here a case study of a protein of interest expressed with different promoter elements in P. pastoris. Distinctive expression patterns for a protein of interest (Fig. 4) secreted in P. pastoris were observed when driven using promoter elements belonging to methanol-inducible, methanol-free, and constitutive conditions/categories.

Fig. 4: Expression of a protein of interest in Pichia pastoris with different inducible and constitutive promoter elements, including promoters from orthologous organisms. AOX1 refers to the methanol inducible promoter. Although, P1 and P2 are methanol inducible, they are also switched on in the absence of methanol, also in the absence of glucose. P3 refers to a constitutive promoter.

At Laurus Bio, carrier proteins and enzymes with varied applications were produced P. pastoris.  One such example is Trypsin, which is available in powder as well as in liquid form (Fig.5).  A pipeline of proteins belonging to different functional classes have been produced or is being evaluated in P. pastoris.

Fig.5: USP Trypsin expressed in Pichia pastoris at Laurus Bio is available in liquid and powder form with applications in cell culture and manufacturing of Insulin/vaccines. For more details, please visit https://laurus.bio/our-products/

Hansenula polymorpha is a thermo-tolerant methylotrophic yeast which has been utilized for commercial production of hormones (human parathyroid hormone,), vaccines (hepatitis B vaccines), biopharmaceuticals (Staphylokinase) etc⁸. This granted GRAS (Generally Recognized as Safe) status for this organism, enables the secretory production of recombinant proteins in a biologically active form, achieving high titres. This capability arises from its ability to thrive within a temperature range of 30°C to 50°C, preventing hyper-glycosylation. Additionally, it facilitates high cell-density fermentation, utilizes strong and inducible promoters, and is free from allergens, toxins, and viral contaminations.

We present here a case study of a protein of interest expressed with different promoter elements in H. polymorpha. Distinctive expression patterns for a protein of interest (Fig.6) secreted in H. polymorpha were observed when driven using promoter elements belonging to methanol-inducible, methanol-free, and constitutive conditions/categories.

Fig. 6: Expression of a protein of interest in Hansenula polymorpha driven by different promoter elements.

We have developed a toolkit comprising of different promoters and accessory proteins for evaluation of expression of protein of interest in H. polymorpha.

Exploring new expression systems

Achieving high yields of recombinant protein expression is critical for microbial precision fermentation-derived products to become cost-competitive and drive market adoption. Consequently, there is a continuous search for alternative heterologous systems capable of expressing proteins at high levels. In addition to bacteria and yeast, filamentous fungi such as Trichoderma reesei are also being explored by numerous researchers for this purpose, although their applicability may vary case by case. One challenge lies in the limited availability of reported literature, necessitating the internal construction of expression vectors for protein expression.

At Laurus Bio, we are actively evaluating Trichoderma reesei as a potential expression host. The probability of identifying strains with high expression levels can be significantly enhanced by incorporating high-throughput screening of clones. This approach complements the testing of different expression elements, offering a robust strategy to achieve optimal protein production. By adding another dimension to our screening process, we aim to revolutionize the efficiency of protein expression, bringing us closer to cost-effective precision fermentation-based solutions.

References

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