课程大纲

COURSE SYLLABUS

课程代码/名称

Course Code/Title

蛋白质工程

Protein Engineering and Molecular Targeting

（

PTME

）

课程性质

Compulsory/Elective

选修课

Elective

课程学分/学时

Course Credit/Hours

3 学分/48 学时

授课语言

Teaching Language

英语 English

授课教师 Instructor(s)

Peter Pimpl

先修要求

Pre-requisites

普通生物学，生命科学概论

General Biology

，

Introduction to Life Science

教学目标 Course Objectives

蛋白质是结合当今生命科学各个方面的关键因素。蛋白质是基础研究和技术应用开发的关键参与者。因

此，工程改造蛋白质的能力是成功开展项目研究并在该领域建立未来事业的关键。

本课程阐明了蛋白质工程中的基本原理和策略，并提供了在活细胞中使用功能性工程蛋白来为基础项目

研究和应用科学开发新的研究工具和实验策略的机会。本课程的内容以实际问题为基础，旨在为学生自

己的研究项目或研究兴趣提供解决问题的方法，而不依赖于特定的模式生物。

学生将学习蛋白质工程学的方法、策略和目标（L1-3）；如何工程化蛋白质以控制细胞的形态和功能

（L4）；了解真核和原核生物中蛋白质合成，加工和转运的分子机制（L5-6），这是成功分析蛋白质/细

胞功能和优化蛋白质表达以最大化重组蛋白质产量的要求（L7）；了解生成特异性抗体以进行蛋白质功

能战略分析的优势（L8-9）；如何生成用于快速克隆多域融合蛋白的个性化载体系统（10）；了解在生

命科学（L11-12）中使用工程化纳体的新机会，以及如何在真核细胞中设计和利用纳体-表位融合蛋白用

于战略研究和生物技术应用（L13-14）。

在与每次主题相对应的研讨课上，学生的口头报告可以加深对主题的理解，每一个主题之后也会着重对

实验方法、实验策略和获得的结果进行简短的讨论。

Proteins are the key players that unite all aspects of today’s Life Sciences. Proteins are crucial key players f

basic research and development of technical appl

ications. The ability to engineer proteins is therefore the key to

successfully perform research projects and to establish a future career in this field.

The course pinpoints fundamental principles and strategies in protein engineering and illustrates the

opportunities

of using functionally engineered proteins in living cells to develop new research tools and experimental strategies

for basic research projects and for applied sciences. The content of the course is illustrated based on real-

life

problems and aims at offering problem-

solving approaches for the students' own research projects or research

interests, independent of specific model organism.

The students will learn about methods, strategies and aims in protein engineering (L1-

3), how to engineer

roteins to control morphology and function of cells (L4), understand the molecular mechanisms of synthesis,

processing and trafficking of proteins in eukaryotic and prokaryotic cells (L5-6) as requirement toward

successful analysis of protein/cellular fun

ctions and the optimization of protein expression to maximize

production yields of recombinant proteins (L7), understand the advantages of generating specific antibodies for

strategic analysis of protein functions (L8-9), how to generate a personalized vec

tor system for fast cloning of

multidomain fusion proteins (10), understand the new opportunities of using engineered nanobodies in Life

Science (L11-12), how to design and utilize nanobody-epitope fusion proteins in eukaryotic cells f

or strategic

research and biotechnological applications (L13-14).

The lecture topics are deepened in accompanying seminars by oral presentations by the students, each of which is

followed by a short discussion with focus on methods, experimental strategies and results obtained.

教学方法 Teaching Methods

Lectures (L) and seminars (S) with oral presentations by students (individual/group)

教学内容 Course Contents

Section 1

Lecture 1: Protein engineering for research and technological applications: Aims and

strategies, rational design, directed evolution & de novo design of proteins, prerequisites

and

consequences.

Section 2

Lecture 2: Tagging proteins for purification, detection and functional analysis: Epitope tags,

enzymatic tags, fluorescent protein tags, interaction tags,

Seminar 1 (L1/2)

Section 3

Lecture 3: Engineering proteins to modify function and to improve stability: Kinetic stability,

thermodynamic stability and process stability. Considerations for preproduction,

production

and postproduction pipelines.

Section 4

Lecture 4: Engineering cellular functions in eukaryotic cells: Design of logical gates for

information processing: NOT. NOR, S-R latch via promotor/transcription factor engineering

to control morphogenetic modules.

Seminar 2 (L3/4)

Section 5

Lecture 5: Protein expression in eukaryotic cells: Principles of synthesis, folding and

processing of proteins in eukaryotic cells, considerations and prerequisites for establishing

protein expression strategies for soluble proteins, type I and type II membrane proteins

and

for multi-spanning membrane proteins.

Section 6

Lecture 6: Protein sorting and transport in eukaryotic cells. Prerequisites for successful

targeting of engineered proteins to intracellular locations. Protein sorting mechanisms

soluble /membrane proteins, receptor transport, vesicle-mediated intracellular

protein

transport. Selective protein targeting strategies.

Seminar 3 (L5/6)

Section 7

Lecture 7: Design and use of quantifiable and fluorescent marker and reporter proteins to

analyse protein-protein interactions and protein function in eukaryotic cells. T

ransient gene

expression systems in eukaryotic cells, transfection methods, protein expression and knock-

down strategies, expression of dominant-negative deletion/substitution mutants and protein-

protein interaction analysis.

Section 8

Lecture 8: Generation of specific polyclonal antibodies. Structure and function of IgG

antibodies, cloning, expression and purification of antigens for immunizations,

characterization of immune sera and antibody specificity.

Seminar 4 (L7/8)

Section 9

Lecture 9: Strategic use of specific antibodies to analyse protein location, function and

protein-protein interactions. Antibody-based analyses: Immune fluorescence microscopy

immune electron microscopy, immune precipitation, co-immune precipitation, SDS-PAGE,

cell identification and cell sorting.

Section 10

Lecture 10: Designing vector systems for fast and efficient generation of N-/C-terminal

fusion proteins for signal-peptide/sorting signal-containing proteins for

designing and

generating complex multidomain fusion proteins.

Seminar 5 (L9/10)

Section 11

Lecture 11: Nanobodies: generation, structure and function of nanobodies. Nanobody-based

applications in research, diagnostics and therapeutics. Engineering strategies to enhance

nanobody-epitope interactions. Strategies for epitope mapping and nanobody-

epitope

interaction analysis for in vivo applications.

Section 12

Lecture 12: Design of Nanobody and epitope-tagged fusion proteins for assembly and

intracellular targeting of proteins complexes in eukaryotic cells by nanobodies-

epitope

interaction. Strategies for post-translational protein labelling, intracellular targeting,

protein

tracing and molecular trapping in eukaryotic cells.

Seminar 6 (L11/12)

Section 13

Lecture 13: Design of dual epitope proteins for assembly of differential nanobody-fusion

protein populations in eukaryotic cells. In vivo crosslinking of membrane

proteins in

eukaryotic cells by dual-epitope linker proteins, design and use of dual-epitope linker

analyze and manipulate protein transport and cellular functions.

Section 14

Lecture 14: Nanobody-based in vivo immunoprecipitation (iVIP) strategies. iVIP systems to

analyze composition and stoichiometry of cytosolic protein complexes

and to manipulate

cellular functions in eukaryotic cells, comparison iVIP vs. FRET-FLIM/IP/Co-IP.

Seminar 7 (L13/14)

Section 15

Review session with questions/answers & conclusions

10.

课程考核

Course Assessment

出勤

10%+

课堂表现

10%+

研讨课表现

30%+

家庭作业

50%

Attendance 10%+Class performance 10%+Seminar performance 30 %+Homework 50%

11.

教材及其它参考资料 Textbook and Supplementary Readings

Textbooks for general reading:

1. Molecular Biology of the Cell, Bruce Alberts, Alexander D. Johnson, Julian Lewis, David M

organ, Martin

Raff, 6

edition 2014.

2. Molecular Cell Biology, Harvey Lodish, Arnold Berk, Chris A. Kaiser, Angelika Amon, Hidde Ploegh, 8

edition 2016.