膜荚黄芪苯丙氨酸解氨酶基因在毕赤酵母中的分泌表达

Expression of Astragalus membranaceus phenylalanine ammonia-lyase gene in Pichia pastoris

通过提取膜荚黄芪叶片总RNA进行反转录合成cDNA,根据GenBank上已发表的膜荚黄芪苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)核酸序列设计引物,从cDNA中扩增到PAL的开放阅读框区段,并将该区段亚克隆到pUCm-T载体上.通过设计与毕赤酵母表达载体pPIC9K具有同源区段的引物,从pUCm-T-PAL上扩增出带同源臂的PAL,利用体外同源重组技术将其克隆到pPIC9K中构建成pPIC9K-PAL表达载体.与传统的酶切连接载体构建方法相比,该方法可避免酶切位点可利用性的限制.将SalⅠ线性化的pPIC9K-PAL电转化至毕赤酵母GS115中,于2.0mg/mL G418抗性平板上进行筛选获得阳性克隆.经1%甲醇诱导120h后,重组菌株GS115/pPIC9K-PAL与负对照相比,其SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)电泳图谱在77.96ku处有1条明显的蛋白带,大小与预期PAL蛋白一致.利用Q-Sepharose FF蛋白纯化柱对总蛋白进行纯化,获得了较纯的苯丙氨酸解氨酶.Bradford法测得纯化后PAL质量浓度为0.08mg/mL,含量占总蛋白的11.54%,最高比活达到4 270U/mg.

L-phenylalanine , as an essential amino acid for human nutrition , is widely used in pharmaceutical and food industries . Using phenylalanine ammonia-lyase （ PAL ,EC 4 .3 .1 .5） to produce L-phenylalanine is one of the major routes . However , most commercial enzymes are extracted from Rhodotorula glutinis , which is time-consuming and over-priced . Therefore , how to efficiently construct the genetic engineering strain to produce PAL is the hot topic . Pichia pastoris is popular in expressing heterologous proteins due to the advantages of low nutritional demands , excellent genetic stability and high-density fermentation . Inserting the heterologous gene into pPIC 9K vectortoachievesecretedexpressionin P.pastorishasbeenreported.However,unlikeothervectors,pPIC9Khas few desirable restriction enzyme cutting sites , which reduces vector construction efficiency when the classical method of digestion and then ligation is adopted . Under this condition , an efficient cloning strategy , independent of digestion and ligation , is required . Homologous recombination in vitro between pPIC9K and gene can settle this problem .Now ,we intend to employ homologous recombination in vitro cloning method to insert the PAL gene into pPIC9K vector to obtain secreted expression in P . pastoris in order to lay the basis for industrial fermentation . First , total RNA extracted from Astragalus membranaceus was used as template for isolating cDNA . Open reading frame （ ORF） of PA L gene was amplified by PCR from cDNA with a pair of primers designed according to the sequence of PA L gene published in the GenBank . Then , ORF was cloned into vector pUCm-T . The transformant was selected to sequence for further analysis of the PA L gene sequence with the help of bioinformatics tools . After that , pPIC9K-PA L was constructed by homologous recombination in vitro . Similarly , the transformant was selected to sequence to investigate the base mutation caused by PCR . Linearized pPIC 9K-PA L by SalⅠ was transformed into P . pastoris GS115 by electroporation . Positive strains were screened on MD and then YPD-G418 plates . The strain resistant to 2.0 mg/mL G418 was selected to express induced by 1% methanol every 24 hours . Supernatant was collected for expression analysis at 0 , 12 , 24 , 48 , 72 , 96 , 120 and 144 h . The recombined PAL protein was purified by Q sepharose fast flow chromatography . Finally , protein concentration was measured with Bradford method and enzyme activity was analysed by measuring the absorption of resultant trans-cinnamic acid at 290 nm . After PA L was subcloned into pUCm-T , the result of sequencing indicated that about 2 200 bp ORF sequence of PA L gene was cloned from A . membranaceus . With the help of bioinformatics methods , it was predicted that PA L encoded a protein which was about 78 ku in molecular mass and 6.04 in pI , containing 718 amino acid residues . Amino acid sequence alignment revealed that the PAL shared 99% identity with PAL from A . membranaceus published in the NCBI . After homologous recombination in vitro between pPIC9K and PAL , the two evidences that about 900 bp fragment which was coincident with the expectation was obtained from the recombinant plasmid pPIC9K-PA L by EcoR Ⅰ double digestion and that furthering analysis of sequencing both suggested that PAL was successfully constructed into pPIC9K without any mutation compared with the first sequencing . After linearized pPIC9K-PA L was transformed into P . pastoris GS115 , colony PCR indicated PA L gene was integrated into the yeast chromosome in contrast to the negative control . The strain resistant to 2.0 mg/mL G418 was selected to express . SDS-PAGE demonstrated that a sharp 78 ku protein which was equal to the predicted value was expressed in the supernatant in contrast to the parent pPIC 9K . By Q sepharose fast flow chromatography , the protein was well purified . The concentration of purified protein was 0.08 mg/mL , accounting for 11.54% of total proteins and the specific activity of GS 115/pPIC9K-PA L-3 was 4 270 U/mg . This study supplies a novel cloning strategy to insert the target gene into pPIC9K vector by homologous recombination in vitro which makes vector construction get rid of the limitation of restriction enzyme cutting site . Meanwhile , the expression of PAL gene in P . pastoris GS115 has laid the foundation for using genetic engineering technology to produce PAL in large scale .