Milton D L, Napier M L, Myers R M, Hardman J K
J Biol Chem. 1986 Dec 15;261(35):16604-15.
A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan synthase. A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the alpha-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagenized in vitro (using nitrous acid, formic acid, hydrazine, and potassium permanganate), and several thousands of mutants have been isolated. Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single amino acid substitutions. Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by lactose. The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein. A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures. The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed. Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha-subunit. An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many amino acid alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide.
为了进一步研究大肠杆菌色氨酸合成酶α亚基的折叠行为，启动了诱变方法。将随机单碱基对饱和诱变程序（迈尔斯，R.M.，勒曼，L.S.，和马尼阿蒂斯，T.（1985年）《科学》229卷，242 - 247页）应用于编码该多肽的trpA基因的亚克隆片段。构建了诱变质粒载体，其中包含trpA基因的三个片段，它们共同编码α亚基约一半的总氨基酸残基。构建这些载体时使得每个trpA片段的每条链都可以被改变。这些trpA片段在体外进行诱变（使用亚硝酸、甲酸、肼和高锰酸钾），并且已经分离出数千个突变体。对包含在前两个trpA片段内（这两个片段涵盖trpA基因的前206个碱基对并编码α亚基的前63个残基）的32个突变体进行了测序。其中，20个（63%）包含单碱基对改变，12个（37%）包含多个改变，并且这些碱基对改变中的17个（53%）导致单个氨基酸替换。将选定的突变trpA片段亚克隆到一个过表达载体中，在该载体中trpA基因由tac启动子控制并且可被乳糖诱导。诱导的动力学和程度表明，诱导22小时后，野生型α亚基占总蛋白的约30%。描述了一种用于α亚基的简单一步纯化程序，通过该程序可以从200毫升完全诱导的培养物中获得15毫克α亚基。诱导突变trpA基因表达突变α亚基，并对其在粗提物中的性质进行了初步检查。在所检查的17个突变蛋白中，大多数的过量表达水平与野生型α亚基相当。对这组突变α亚基的表观稳定性、β2亚基激活活性和内在活性的分析表明，许多氨基酸改变没有明显影响；存在多种新的功能缺陷；并且位于残基28至54附近的一个序列对于该多肽的正常折叠可能特别关键。
