荧光假单胞菌蛋白表达系统研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2024-0506

摘要/Abstract

摘要：

荧光假单胞菌（Pseudomonas fluorescens）作为一种新型蛋白表达系统，凭借其多样且独特的分泌系统，逐渐展现出在生物技术和生物制药领域的巨大应用潜力。尽管传统蛋白表达系统，如大肠杆菌、酿酒酵母及昆虫细胞系统，已在众多研究和工业应用中取得成功，但仍存在折叠机制和翻译后修饰等方面的局限性。相较之下，荧光假单胞菌因其能够高效处理和分泌大分子蛋白质而迅速成为当前研究的热点。本文综述了荧光假单胞菌在蛋白表达系统方面的最新进展，涵盖了高效表达宿主菌株、克隆表达载体及调控元件、蛋白分泌系统等多个领域。详细分析了荧光假单胞菌在蛋白质折叠、分泌及糖基化修饰等关键步骤中的优异表现，并探讨了多种信号肽在优化蛋白分泌效率中的应用。这些研究成果不仅显著提升了目标蛋白的溶解度和表达水平，还为改善蛋白质生产工艺提供了新的思路。此外，荧光假单胞菌在疫苗、治疗性蛋白质和抗体生产中的应用也已显示出广阔的应用前景。尽管近年来的研究取得了显著进展，该系统在某些高分子蛋白表达方面仍面临挑战。未来的研究应集中于基因组编辑技术、蛋白质分泌调控机制的优化，以及工业生产中的工艺优化，以进一步提高荧光假单胞菌作为蛋白表达平台的灵活性和效率，为蛋白质工程和合成生物学提供强有力的工具。

关键词: 荧光假单胞菌, 蛋白表达系统, 分泌系统

Abstract:

Pseudomonas fluorescens, as an emerging protein expression system, has demonstrated significant application potential in the fields of biotechnology and biopharmaceuticals, due to its diverse and unique secretion systems. While traditional protein expression systems such as Escherichia coli, Saccharomyces cerevisiae, and insect cell systems have achieved considerable success in many research and industrial applications, they still exhibit limitations in protein folding mechanisms and post-translational modifications. In contrast, P. fluorescens has rapidly gained attention as a research hotspot for its ability to efficiently process and secrete large, complex proteins. This review summarizes recent advances in P. fluorescens as a protein expression system, covering areas such as efficient host strains, cloning vectors, regulatory elements, and protein secretion systems. The paper provides a detailed analysis of P. fluorescens's performance in key steps like protein folding, secretion, and glycosylation, while also exploring the application of various signal peptides in optimizing protein secretion efficiency. These findings not only significantly enhance the solubility and expression levels of target proteins but also offer new insights into improving protein production processes. Furthermore, the application of P. fluorescens in vaccine, therapeutic protein, and antibody production has shown promising potential. Despite significant progress in recent years, challenges remain in expressing certain high-molecular-weight proteins. Future research should focus on genome editing technologies, optimization of protein secretion regulation, and improvements in industrial production processes to further enhance the flexibility and efficiency of P. fluorescens as a protein expression platform, providing a powerful tool for protein engineering and synthetic biology.

Key words: Pseudomonas fluorescens, protein expression system, secretion system

谭景轩, 邢德勋, 何天锦, 刘占英. 荧光假单胞菌蛋白表达系统研究进展[J]. 生物技术通报, 2025, 41(1): 49-61.

TAN Jing-xuan, XING De-xun, HE Tian-jin, LIU Zhan-ying. Advances in Protein Expression System of Pseudomonas fluorescens[J]. Biotechnology Bulletin, 2025, 41(1): 49-61.

图/表 5

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表达载体 Expression vector	复制子 Replicon	启动子 Promoter	蓝白斑筛选 Blue-white screening	诱导物 Inducer	抗性标记 Resistance marker	特征 Characteristics	参考文献 Reference
pCN51	pPS10	P_tac, P_lac	No	IPTG by P_lac	Kana	适用于多种假单胞菌	[32]
pBBR1MCS	pBBR1	P_tac, P_lac	Yes	IPTG by P_lac	Cm	适用多种启动子系统	[33]
pEX18Gm	pEX18	lacZ	Yes	IPTG	Gm	基因敲除和替换载体	[34]
pQF50	ColE1	lacZ	Yes	IPTG	Amp	基于lac启动子系统	[35]
pET series	ColE1	P_T7	No	IPTG	Amp	需配合T7 RNA聚合酶	[36]
pEB8	RSF1010	P_T7	No	IPTG	Amp	适用于含T7 RNA聚合酶的宿主菌株	[37]
pPLGN1	RSF1010	λ_PR	No	Temperature	Kana	温度调控表达	[38]
pLV vectors	RSF1010	λ_PL	No	Temperature	Kana	温度调控表达	[39]
pERD20/pERD21	RSF1010	P_m	No	Benzoate	Kana	苯甲酸诱导	[40]
pVLT vectors	RSF1010	P_tac	No	IPTG	Kana	IPTG诱导表达	[41]
pMMB66EH	RSF1010	P_tac	No	IPTG	Amp	IPTG诱导表达	[42]
pJB653 and derivatives	RK2	P_m	No	m-Toluic acid	Kana	m-甲苯酸诱导	[43]
pRK415	RK2	P_lac	Yes	IPTG	Tet	具有蓝白筛选功能	[44]
pUCP vectors	pRO1600	P_lac	Yes	IPTG	Carb	具有蓝白筛选功能	[45]
pUCPKS/pUCPSK	pRO1600	P_T7, P_lac	Yes	IPTG	Carb	双启动子系统	[46]
pBSP II KS/pBSP II KS	pRO1600	P_T7, P_lac	Yes	IPTG	Amp	双启动子系统	[47]

表达载体 Expression vector	复制子 Replicon	启动子 Promoter	蓝白斑筛选 Blue-white screening	诱导物 Inducer	抗性标记 Resistance marker	特征 Characteristics	参考文献 Reference
pCN51	pPS10	P_tac, P_lac	No	IPTG by P_lac	Kana	适用于多种假单胞菌	[32]
pBBR1MCS	pBBR1	P_tac, P_lac	Yes	IPTG by P_lac	Cm	适用多种启动子系统	[33]
pEX18Gm	pEX18	lacZ	Yes	IPTG	Gm	基因敲除和替换载体	[34]
pQF50	ColE1	lacZ	Yes	IPTG	Amp	基于lac启动子系统	[35]
pET series	ColE1	P_T7	No	IPTG	Amp	需配合T7 RNA聚合酶	[36]
pEB8	RSF1010	P_T7	No	IPTG	Amp	适用于含T7 RNA聚合酶的宿主菌株	[37]
pPLGN1	RSF1010	λ_PR	No	Temperature	Kana	温度调控表达	[38]
pLV vectors	RSF1010	λ_PL	No	Temperature	Kana	温度调控表达	[39]
pERD20/pERD21	RSF1010	P_m	No	Benzoate	Kana	苯甲酸诱导	[40]
pVLT vectors	RSF1010	P_tac	No	IPTG	Kana	IPTG诱导表达	[41]
pMMB66EH	RSF1010	P_tac	No	IPTG	Amp	IPTG诱导表达	[42]
pJB653 and derivatives	RK2	P_m	No	m-Toluic acid	Kana	m-甲苯酸诱导	[43]
pRK415	RK2	P_lac	Yes	IPTG	Tet	具有蓝白筛选功能	[44]
pUCP vectors	pRO1600	P_lac	Yes	IPTG	Carb	具有蓝白筛选功能	[45]
pUCPKS/pUCPSK	pRO1600	P_T7, P_lac	Yes	IPTG	Carb	双启动子系统	[46]
pBSP II KS/pBSP II KS	pRO1600	P_T7, P_lac	Yes	IPTG	Amp	双启动子系统	[47]

信号肽 Signal peptide	来源 Origin	功能 Function	应用实例 Application	表达特性 Characteristics	参考文献 Reference
PelB	枯草芽胞杆菌的果胶酶B	引导重组蛋白通过Sec途径分泌至周质空间	G-CSF（人粒细胞集落刺激因子）	可溶，活性，96孔板规模为250 mg/L	[59]
OmpA	大肠杆菌的外膜蛋白A	引导外膜蛋白的分泌	G-CSF（人粒细胞集落刺激因子）	可溶，活性，96孔板规模为250 mg/L	[59]
PhoA	大肠杆菌的碱性磷酸酶	引导碱性磷酸酶的分泌	Fab	可溶，活性，100 mg/L	[60]
TorA	大肠杆菌的甲基氨基甲酸酯还原酶	专用于Tat途径，适合复杂酶类的分泌	人血清白蛋白（HSA）	可溶，活性，1.5 g/L	[61]
LipA	荧光假单胞菌的脂肪酶	引导脂肪酶的分泌	重组脂肪酶（recombinant lipase）	可溶，活性，100 mg/L	[62]
AmyL	枯草芽胞杆菌的α-淀粉酶	引导α-淀粉酶的分泌	重组α-淀粉酶	可溶，活性，85 g/L	[51]
DsbA	大肠杆菌的二硫键异构酶	本身是一种二硫键异构酶，帮助新合成蛋白形成正确的二硫键	人类生长激素（hGH）	可溶，活性，1.2 g/L	[63]
HlyA	大肠杆菌的血红素结合蛋白	通过I型分泌系统引导分泌血红素结合蛋白	血红素结合蛋白	可溶，活性，产量未知	[64]
AprA	荧光假单胞菌的碱性蛋白酶	引导碱性蛋白酶的分泌	碱性蛋白酶	可溶，活性，产量未知	[65]

信号肽 Signal peptide	来源 Origin	功能 Function	应用实例 Application	表达特性 Characteristics	参考文献 Reference
PelB	枯草芽胞杆菌的果胶酶B	引导重组蛋白通过Sec途径分泌至周质空间	G-CSF（人粒细胞集落刺激因子）	可溶，活性，96孔板规模为250 mg/L	[59]
OmpA	大肠杆菌的外膜蛋白A	引导外膜蛋白的分泌	G-CSF（人粒细胞集落刺激因子）	可溶，活性，96孔板规模为250 mg/L	[59]
PhoA	大肠杆菌的碱性磷酸酶	引导碱性磷酸酶的分泌	Fab	可溶，活性，100 mg/L	[60]
TorA	大肠杆菌的甲基氨基甲酸酯还原酶	专用于Tat途径，适合复杂酶类的分泌	人血清白蛋白（HSA）	可溶，活性，1.5 g/L	[61]
LipA	荧光假单胞菌的脂肪酶	引导脂肪酶的分泌	重组脂肪酶（recombinant lipase）	可溶，活性，100 mg/L	[62]
AmyL	枯草芽胞杆菌的α-淀粉酶	引导α-淀粉酶的分泌	重组α-淀粉酶	可溶，活性，85 g/L	[51]
DsbA	大肠杆菌的二硫键异构酶	本身是一种二硫键异构酶，帮助新合成蛋白形成正确的二硫键	人类生长激素（hGH）	可溶，活性，1.2 g/L	[63]
HlyA	大肠杆菌的血红素结合蛋白	通过I型分泌系统引导分泌血红素结合蛋白	血红素结合蛋白	可溶，活性，产量未知	[64]
AprA	荧光假单胞菌的碱性蛋白酶	引导碱性蛋白酶的分泌	碱性蛋白酶	可溶，活性，产量未知	[65]

分泌系统 Secretion system	膜锚定位置Membrane anchoring site		存在种属Species present				参考文献 Reference
分泌系统 Secretion system	IM	OM	G^-	G⁺	Arch	Euk	参考文献 Reference
Sec secretory pathway	+	-	+	+	+	+	[54]
Twin arginine targeting（Tat）pathway	+	-	+	+	+	+	[66]
Holins	+	-	+	+	+	-	[72]
Large conductance mechanosensitive ion channel（MscL）	+	-	+	+	+	+	[73]
Outer membrane FUP	-	+	+	+	-	-	[74]
Type I secretion system（ABC）	+	-	+	+	-	+	[67]
Type II secretion system（MTB）	+	-	+	+	-	-	[67]
Type III secretion system	+	-	+	-	-	-	[67]
Type V secretion system（AT）	+	-	+	-	-	-	[67]
Type VI secretion system	+	-	+	-	-	-	[68]