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首页> 外文期刊>Applied Microbiology and Biotechnology >Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis
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Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis

机译：基于pKD1的质粒系统对乳酸克鲁维酵母中异源蛋白质生产的评估

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The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gall-209 K. lactis strain was employed. Selective medium containing 5 g glucose/1 and 0.5 g galactose (inducer)/1 allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular P-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the P-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial P-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pK1 vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times. [References: 24]
机译：在酵母克鲁维酵母中细胞内和细胞外基因产物的合成过程中评估了基于pKD1的载体的稳定性。将大肠杆菌lacZ和MFalpha1前导肽BPTI（牛胰胰蛋白酶抑制剂）盒置于可诱导的乳酸克鲁维酵母LAC4启动子的控制下，并插入基于pKD1的质粒中。为了在维持诱导剂水平的同时诱导基因表达，使用了免费的gal-209乳酸乳球菌菌株。含有5 g葡萄糖/ 1和0.5 g半乳糖（诱导剂）/ 1的选择性培养基可实现异源产物的最佳表达和分泌，而对重组细胞的生长无明显影响。在长期连续分批培养（60代）中，质粒不稳定主要是结构不稳定的结果。相对于细胞内P-半乳糖苷酶，BPTI的表达和分泌导致更大的结构不稳定性。对于这两种产品，携带pKD1复制起点和顺式作用稳定性位点的载体（部分pKD1载体）比携带完整pKD1序列的载体（完整pKD1载体）更稳定。然而，经过55代后，全pKD1载体的P-半乳糖苷酶和BPTI活性仍然更高。这是由于相对于部分pK1载体，全pKD1载体的初始P-半乳糖苷酶和BPTI活性明显较高（分别高出约85％和47％）。 Southern印迹证实这些增加是由于携带完整pKD1序列的载体的拷贝数更高。与我们先前报道的转化酶分泌结果相反，全pKD1载体至少在55代内被用于表达/分泌β-半乳糖苷酶和BPTI。由于其结构稳定性，部分pKD1载体对于非常长的培养时间将是有利的。 [参考：24]

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