未折叠蛋白反应激活因子基因HAC1P的共表达促进毕赤酵母茎菠萝蛋白酶的产生

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-08-20 DOI:10.1016/j.pep.2025.106804

Sindhu Varadharaj , Jayachandran Krishnan , Mohd Imran Shah , Meenakshisundaram Sankaranarayanan

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引用次数: 0

摘要

高滴度表达外源蛋白和代谢物会对重组宿主产生多种应激反应。菠萝蛋白酶是一种半胱氨酸蛋白酶，存在于菠萝植物Ananas comosus的茎和果实中。该酶具有广泛的工业应用范围，从食品，保健品，化妆品和制药。本研究旨在研究未折叠蛋白反应调节因子（UPR） Hac1对毕氏酵母中重组茎菠萝蛋白酶折叠和分泌的影响。利用组成型启动子和诱导型启动子在毕赤酵母X-33宿主中进行了菠萝蛋白酶基因（BL）的密码子优化和表达。为了折叠UPR调控因子内质网（ER）中错误折叠的蛋白聚集体，Hac1p在GAP启动子的组成表达下与菠萝蛋白酶共表达。摇瓶研究结果表明，当HAC1与茎菠萝蛋白酶（BL）共表达时，蛋白酶活性增加了2倍，为4 U/mL。在3.7 L KLF生物反应器中，在甘油限制条件下，对pgapap α bl1和pGAPBLHAC1克隆进行了批量投料研究，最高活性分别为8 U/mL和54 U/mL。折叠和分泌通路主要基因的基因表达研究表明，UPR的激活导致Kar2p、Sec 63、Pdi、Cne1等主要伴侣蛋白上调。茎菠萝蛋白酶活性为54 U/mL，是迄今为止文献报道的最高活性。目前的工作表明毕赤酵母作为一个强大的平台生产茎菠萝蛋白酶用于各种工业应用。

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Enhanced production of stem bromelain in Pichia pastoris by coexpression of unfolded protein response activator gene HAC1P

Expression of heterologous proteins and metabolites at high titers mounts several stress responses on the recombinant host. Stem Bromelain is a cysteine protease enzyme present in the stem and fruit of the pineapple plant Ananas comosus. The enzyme has a broad range of industrial application ranging from food, nutraceutical, cosmetic and pharmaceutical. The current work aims to study the effect of unfolded protein response regulator (UPR) Hac1 on folding and secretion of recombinant stem bromelain in Pichia pastoris. Stem bromelain gene (BL) from Ananas comosus was codon optimized and expressed in the Pichia pastoris X-33 host using constitutive and inducible promoters. To fold the misfolded protein aggregates in Endoplasmic Reticulum (ER), UPR regulator, Hac1p was co-expressed with bromelain under constitutive expression by GAP promoter. Shake flask studies resulted in a 2-fold increase in the protease activity of 4 U/mL when HAC1 was co-expressed with stem bromelain (BL). Fed batch studies were performed for both pGAPαBL1 and pGAPBLHAC1 clones in 3.7 L KLF bioreactor under glycerol limited condition and highest activity of 8 U/mL and 54 U/mL were obtained respectively. Gene expression studies of the major genes in folding and secretion pathway has shown that the activation of UPR has resulted in upregulation of major chaperones like Kar2p, Sec 63, Pdi, Cne1. The stem bromelain activity of 54 U/mL is the highest activity reported so far in the literature. The current work signifies Pichia pastoris as a robust platform to produce stem bromelain for various industrial applications.

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.