4、Client Case Studies

Case 1:

We successfully completed the expression and purification of a CRISPR-Cas protein targeting tRNA for a client. This protein is a novel CRISPR effector protein capable of specifically recognizing and cleaving tRNA molecules, holding significant application potential in gene regulation and epigenetic research. The client required a high-purity, active protein with native conformation for subsequent functional validation experiments. However, this protein was prone to forming inclusion bodies when expressed in prokaryotic systems and was sensitive to buffer conditions, making purification challenging. To address these challenges, we achieved soluble expression through codon optimization and expression condition screening, and successfully obtained the target protein with purity exceeding 95% using a multi-step purification strategy combining affinity chromatography and ion exchange chromatography. The client's activity assays confirmed that the protein exhibited specific tRNA cleavage activity, fully meeting the requirements for subsequent cellular experiments. The client highly recognized our professional expertise, problem-solving capabilities, and efficient delivery.

Case 2:

We successfully assisted a client in achieving soluble expression and purification of a membrane protein, overcoming a long-standing bottleneck that had hindered their research.

The client was studying a structurally complex membrane protein with important biological functions, playing a key role in signal transduction. The client needed high-purity, active protein with native conformation for structural biology studies and drug screening. However, the inherent hydrophobicity of membrane proteins makes them prone to forming inactive aggregates in conventional expression systems. Moreover, once removed from their native membrane environment, protein stability sharply declines, and purification is often accompanied by loss of function—this had become the core technical barrier impeding the client's research progress.

To address this challenge, we first performed sequence analysis of the target membrane protein's transmembrane and hydrophilic regions. We then carried out protein engineering—by fusing specific solubilization tags, optimizing hydrophobic residues in the transmembrane domains, and screening suitable homologous host expression systems—which significantly enhanced the soluble expression level of the protein. Building on this, we systematically screened a variety of mild detergents and established a complete purification workflow encompassing affinity capture, detergent exchange, and fine polishing via size exclusion chromatography, ensuring that the protein remained stably folded throughout the purification process.

Ultimately, we successfully obtained milligram quantities of the membrane protein sample with high purity and correct conformation. The client's functional validation confirmed that the sample retained the expected biological activity and could be directly used for subsequent crystallization screening and small molecule binding assays. The client highly recognized our technical expertise and customized solution in the challenging field of membrane protein purification.

Case 3:

We successfully completed the expression and purification of a highly toxic methyltransferase protein for a client, providing a critical reagent for their epigenetics research.

The client was studying a novel methyltransferase involved in histone modification, which plays a central regulatory role in gene silencing and chromatin remodeling. The client needed high-purity enzyme protein with native catalytic activity for in vitro activity assays and inhibitor screening. However, this methyltransferase exhibited strong cytotoxicity towards commonly used expression hosts such as E. coli. Under conventional induction conditions, host cell growth was severely inhibited or even lysed, resulting in extremely low expression levels. Additionally, the enzyme showed poor stability during purification, being highly prone to degradation or aggregation, making the preparation of active protein samples a formidable challenge.

To address this highly difficult challenge, we first adopted a tightly regulated expression vector combined with low-temperature culture and reduced inducer concentrations to minimize the metabolic burden of protein expression on the host. This strategy successfully achieved soluble expression while maintaining normal cell growth. Subsequently, we further enhanced protein folding efficiency and stability by co-expressing molecular chaperones and optimizing culture medium components. For the purification step, we designed a gentle, rapid purification process, adding specific cofactors and protective agents throughout the procedure, and combined affinity chromatography with size exclusion chromatography to obtain high-purity samples while preserving enzymatic activity.

Ultimately, we successfully produced milligram quantities of the methyltransferase protein with purity exceeding 90% and robust enzymatic activity. The client's subsequent enzyme kinetic assays confirmed that the protein activity fully met expectations and could be used directly for screening candidate compounds. The client highly appreciated our professional expertise, precise control over toxic protein expression, and efficient solutions.