Fazen Christopher H, Kahkoska Anna R, Doyle Robert P
Department of Chemistry, Center for Science and Technology, Syracuse University, Syracuse, NY 13244, USA.
Protein Expr Purif. 2012 Sep;85(1):51-9. doi: 10.1016/j.pep.2012.06.015. Epub 2012 Jul 3.
Human PYY(3-36) (hPYY3-36) is a 34 amino acid hormone that has received a great deal of attention due to its effects on appetite regulation. hPYY(3-36) was modified at the N-terminus with an octahistidine tag and factor Xa protease sequence along with the small ubiquitin-like modifier (SUMO) tag and expressed in Escherichia coli. The protein was purified from clarified E. coli lysate by immobilized metal affinity chromatography (IMAC) with a yield of 30±7 mg/L of induced culture returned as an average over seven runs, and its identity was confirmed by Western blot and hPYY antibody recognition. The SUMO-tagged hPYY(3-36) was digested with two different proteases to return either His-tagged hPYY(3-36) or unmodified hPYY(3-36): (1) digestion with SUMO protease proceeded at about 50% efficiency yielding His-tagged hPYY(3-36); (2) digestion with factor Xa protease proceeded at greater than 90% efficiency yielding final hPYY(3-36). Products were purified from the digestion mixtures by reverse-phase high-performance liquid chromatography (C(18)) or IMAC, respectively, the identities were confirmed by mass spectrometry and hPYY antibody recognition, and the folded state of His-tagged hPYY(3-36) was investigated by circular dichroism spectroscopy.
人源PYY(3 - 36)（hPYY3 - 36）是一种由34个氨基酸组成的激素，因其对食欲调节的作用而备受关注。hPYY(3 - 36)在N端被八组氨酸标签、因子Xa蛋白酶序列以及小泛素样修饰物（SUMO）标签修饰，并在大肠杆菌中表达。通过固定化金属亲和色谱（IMAC）从澄清的大肠杆菌裂解物中纯化该蛋白，七次运行的平均诱导培养物产量为30±7 mg/L，其身份通过蛋白质印迹法和hPYY抗体识别得以确认。用两种不同的蛋白酶消化SUMO标签化的hPYY(3 - 36)，以得到带His标签的hPYY(3 - 36)或未修饰的hPYY(3 - 36)：（1）用SUMO蛋白酶消化的效率约为50%，产生带His标签的hPYY(3 - 36)；（2）用因子Xa蛋白酶消化的效率大于90%，产生最终的hPYY(3 - 36)。分别通过反相高效液相色谱（C(18)）或IMAC从消化混合物中纯化产物，通过质谱分析和hPYY抗体识别确认其身份，并通过圆二色光谱研究带His标签的hPYY(3 - 36)的折叠状态。
