Multiple strategies to improve the yield of snake thrombin-like enzyme agkihpin from Gloydius halys pallas in Pichia pastoris

Background In our previous research, we successfully identified agkihpin, a snake venom thrombin-like enzyme (SVTLE) from Gloydius halys pallas . It possesses thrombin-like activity and exhibits multiple functions, including inhibition of tumor invasion and metastasis. Its unique enzymatic properties make agkihpin a promising candidate for thrombolytic and anti-metastatic therapies. In an earlier study, we attempted to produce agkihpin recombinantly in Escherichia coli ( E. coli ). However, the protein was predominantly expressed as inclusion bodies, with low solubility and negligible enzymatic activity before in vitro refolding. Results In this study, we developed a multi-strategy approach in Pichia pastoris (reclassified as Komagataella phaffii [1]; herein P. pastoris ) to enhance the secretory expression of agkihpin. This approach mainly included gene dosage optimization, N-glycosylation engineering, and co-expression of helper factor proteins. In the absence of helper factor proteins, the recombinant strain carrying two copies of agkihpin exhibited the highest level of expression. N-glycosylation plays a major role in maintaining expression levels in this system, as removal of the glycosylation site reduced the yield to approximately 18.6% of the wild-type secretion level. Among the 11 helper factor proteins evaluated, Bmh2, a protein involved in the secretory pathway, significantly increased agkihpin yield by approximately 68% (P < 0.05). Conclusion Studies on helper factor protein co-expression for snake venom protein production in yeast remain scarce. Given the disulfide-rich nature of these proteins, early engineering strategies have predominantly concentrated on oxidative folding enhancement. Our findings, however, suggest that ER-to-Golgi trafficking may also represent a bottleneck during agkihpin expression in P. pastoris , as facilitating this step improves protein secretion. Although validated so far only for a single snake venom protein, this finding suggests that secretory pathway engineering and oxidative folding can synergize to boost titers of recombinant toxins in P. pastoris , a principle that may apply more broadly.