要求目的蛋白中不能含有信号肽酶切割序列：glu-lys-arg-glu-ala-glu-ala。信号肽的切除分为两步：kex2在glu-lys-arg和glu-ala-glu-ala之间初步切除，STE123在两个glu-ala之间切割。
以pPIC9K为例，pPIC9K是用于在毕赤酵母中的多拷贝整合分泌表达蛋白的穿梭载体，利用组氨酸缺陷型标记进行互补筛选。
一、信号肽的2步切割
pPIC9K载体带有自身信号肽，在分泌过程中被切除，但是由于切割效率，在目的产物N端带有3－5个左右的信号肽的氨基酸。
设计引物时，为了防止切割不完全，能去掉GluAla两个重复单位，不过文献报道多个Glu会提高kex2的切割效率。
要求目的蛋白中不能含有信号肽酶切割序列：glu-lys-arg-glu-ala-glu-ala。
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信号肽的切除分为两步：kex2在glu-lys-arg和glu-ala-glu-ala之间初步切除，STE123在两个glu-ala之间切割。
二、是否在3' 端是带上一些其本来的序列？
1,首先，你要保证表达后细胞内信号肽酶能正确识别原来的位点，即此位点是不能随便增减的，否则信号肽不能正常切割，在这个基础上你再决定3' 端其本来序列的去留。
2，一般5'端多出一些氨基酸不会明显影响到你的目的蛋白的活性，我们平时在做酵母表达
时，信号肽切割后，目的蛋白往往有几个多余的AA，这对活性好象没什么影响，虽然如此，但你还得注意多余AA的性质，不能过酸或过碱。
三、重组pPIC9K/GS115表达出目的条带后，
确定信号肽是否切除完全的方法：
1、N端测序
2、先查查你的氨基酸序列里有没有糖基化位点，如果有就只能N端测序了。如果没有，看分子量大小。
信号肽中有kex2和ste13两个酶切位点，现在担心的是kex2的位点酶切完全了，但ste13的两个酶切位点glu， ala重复序列没有切干净，如果这两个氨基酸没有切除完全的话，分子量差异不大，两个氨基酸的差异应该看不出来，一般来说，N端多一两个AA应该对蛋白的活性没有太大影响。western检测有两个条带的问题，可能是目的蛋白有一部分被降解了。
四、附：信号肽切割原理和优化切割深处作文
《Expression of Your Recombinant Protein with a Native N-terminus》
If you wish to have your protein expresd with a native N-terminus, you should clone
your gene flush(直接地) with the Kex2 cleavage site. U PCR to rebuild the quence from the Xho I site at bp 1184-1189 to the arginine codon at nucleotides 1193-1195. Remember to include the first amino acid of your protein, if necessary, for correct fusion to the Kex2 cleavage site.
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Signal Sequence Processing
The processing of the α-factor signal quence in pPICZα occurs in two steps:
1. The preliminary cleavage of the signal quence by the KEX2 gene product,
final Kex2 cleavage occurring between arginine and glutamine in the quence
Lys-Arg * Glu-Ala-Glu-Ala, where * is the site of cleavage.
2. The Glu-Ala repeats are further cleaved by the STE13 gene product.
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Optimization of Signal Cleavage
In Saccharomyces cerevisiae, it has been noted that the Glu-Ala repeats are not necessary
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for cleavage by Kex2, but cleavage after Glu-Lys-Arg may be more efficient when
followed by Glu-Ala repeats. A number of amino acids are tolerated at site X instead of
Glu in the quence Glu-Lys-Arg-X. The amino acids include the aromatic amino acids,
small amino acids, and histidine. Proline, however, will inhibit Kex2 cleavage. For more
飘落的枫叶information on Kex2 cleavage, plea e (Brake et al., 1984).
There are some cas where Ste13 cleavage of Glu-Ala repeats is not efficient, and Glu-
Ala repeats are left on the N-terminus of the expresd protein of interest. This is
generally dependent on the protein of interest.
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